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Insufficient information is available regarding the administration of anticoagulants, specifically direct oral anticoagulants, in individuals with cirrhosis awaiting liver transplantation. In this report, we present a case of a 66-year-old male with atrial fibrillation treated with dabigatran who received idarucizumab prior to orthotopic liver transplantation. Hemostatic status was monitored throughout the procedure with both conventional hemostatic tests and point-of-care viscoelastic hemostatic assays. The patient suffered an intraoperative myocardial infarction, which could be related to the use of idarucizumab.
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Plastics pose a health hazard to living beings and the environment. Plastic degradation produces nano-sized plastic particles (NPs) that end up in terrestrial and aquatic ecosystems, including oceans, rivers, and lakes. Their presence in air, drinking water, sediments, food, and personal care products leads to a variety of exposure routes for living beings, including humans. The toxicity mechanisms of these nanomaterials (NMs) in living organisms and ecosystems are currently unknown, making it a priority to understand their effects at the molecular and cellular levels. The zebrafish (Zf) (Danio rerio) is a model organism which has a high homology with humans and has been widely used to assess the hazard of different xenobiotics. In this study, the expression changes of different genes in 120 hpf Zf embryos (Zfe) after exposure to polystyrene (PS) NPs (30 nm) at concentrations of 0.1, 0.5 and 3 ppm were investigated. The results showed that the gene encoding heat shock protein (hsp70) was down-regulated in a dose-dependent manner. The genes encoding superoxide dismutase (SOD 1 and SOD 2), apoptotic genes (cas 1 and cas 8) and interleukin 1-ß (il1ß) were activated at the concentration of 3 ppm PS NP, while the anti-apoptotic gene Bcl2α was inhibited at 0.5 and 3 ppm. In addition, the neurotransmitter-related gene Acetyl-Cholinesterase (ache) was significantly inhibited and the DNA repair genes (gadd45α and rad51) were also down-regulated. In contrast, the mitochondrial metabolism-related gene cox1 did not alter its expression in any of the treatments. Most of the changes in gene expression occurred at the highest concentration of NPs. Overall, the results indicated that NPs generated cellular stress that caused certain alterations in normal gene expression (oxidative stress, apoptotic and inflammatory processes, neurotoxicity and anti-apoptotic proteins), but did not cause any mortality after 120 hpf exposure at the three concentrations assayed. These results highlight the need for further studies investigating the effects, at the molecular level, of these materials in humans and other living organisms.
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Poluentes Químicos da Água , Peixe-Zebra , Animais , Ecossistema , Microplásticos/toxicidade , Plásticos/toxicidade , Poliestirenos/toxicidade , Poliestirenos/metabolismo , Superóxido Dismutase/genética , Poluentes Químicos da Água/toxicidade , Poluentes Químicos da Água/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Fetal undernutrition programs cardiometabolic diseases, with higher susceptibility in males. The mechanisms implicated are not fully understood and may be related to sex differences in placental adaptation. To evaluate this hypothesis, we investigated placental oxidative balance, vascularization, glucocorticoid barrier, and fetal growth in rats exposed to 50% global nutrient restriction from gestation day 11 (MUN, n = 8) and controls (n = 8). At gestation day 20 (G20), we analyzed maternal, placental, and fetal weights; oxidative damage, antioxidants, corticosterone, and PlGF (placental growth factor, spectrophotometry); and VEGF (vascular endothelial growth factor), 11ß-HSD2, p22phox, XO, SOD1, SOD2, SOD3, catalase, and UCP2 expression (Western blot). Compared with controls, MUN dams exhibited lower weight and plasma proteins and higher corticosterone and catalase without oxidative damage. Control male fetuses were larger than female fetuses. MUN males had higher plasma corticosterone and were smaller than control males, but had similar weight than MUN females. MUN male placenta showed higher XO and lower 11ß-HSD2, VEGF, SOD2, catalase, UCP2, and feto-placental ratio than controls. MUN females had similar feto-placental ratio and plasma corticosterone than controls. Female placenta expressed lower XO, 11ß-HSD2, and SOD3; similar VEGF, SOD1, SOD2, and UCP2; and higher catalase than controls, being 11ß-HSD2 and VEGF higher compared to MUN males. Male placenta has worse adaptation to undernutrition with lower efficiency, associated with oxidative disbalance and reduced vascularization and glucocorticoid barrier. Glucocorticoids and low nutrients may both contribute to programming in MUN males.
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Desenvolvimento Fetal , Feto/metabolismo , Desnutrição/complicações , Fenômenos Fisiológicos da Nutrição Materna , Fator de Crescimento Placentário/metabolismo , Placenta/metabolismo , Caracteres Sexuais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Peso Corporal , Feminino , Peroxidação de Lipídeos , Masculino , Desnutrição/sangue , Oxirredução , Gravidez , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismoRESUMO
One of the consequences of high altitude (hypobaric hypoxia) exposure is the development of right ventricular hypertrophy (RVH). One particular type of exposure is long-term chronic intermittent hypobaric hypoxia (CIH); the molecular alterations in RVH in this particular condition are less known. Studies show an important role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex-induced oxidative stress and protein kinase activation in different models of cardiac hypertrophy. The aim was to determine the oxidative level, NADPH oxidase expression and MAPK activation in rats with RVH induced by CIH. Male Wistar rats were randomly subjected to CIH (2 days hypoxia/2 days normoxia; n = 10) and normoxia (NX; n = 10) for 30 days. Hypoxia was simulated with a hypobaric chamber. Measurements in the RV included the following: hypertrophy, Nox2, Nox4, p22phox, LOX-1 and HIF-1α expression, lipid peroxidation and H2O2 concentration, and p38α and Akt activation. All CIH rats developed RVH and showed an upregulation of LOX-1, Nox2 and p22phox and an increase in lipid peroxidation, HIF-1α stabilization and p38α activation. Rats with long-term CIH-induced RVH clearly showed Nox2, p22phox and LOX-1 upregulation and increased lipid peroxidation, HIF-1α stabilization and p38α activation. Therefore, these molecules may be considered new targets in CIH-induced RVH.
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Regulação Enzimológica da Expressão Gênica , Hipertrofia Ventricular Direita/enzimologia , Hipóxia/enzimologia , Sistema de Sinalização das MAP Quinases , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , NADPH Oxidase 2/biossíntese , Regulação para Cima , Animais , Doença Crônica , Modelos Animais de Doenças , Hipertrofia Ventricular Direita/etiologia , Hipertrofia Ventricular Direita/patologia , Hipóxia/complicações , Hipóxia/patologia , Masculino , Ratos , Ratos WistarRESUMO
Lifestyle and genetic background are well known risk factors of cardiovascular disease (CVD). A third contributing factor is suboptimal fetal development, due to nutrient or oxygen deprivation, placental insufficiency, or exposure to toxic substances. The fetus adapts to adverse intrauterine conditions to ensure survival; the immediate consequence is low birth weight (LBW) and the long-term effect is an increased susceptibility to develop CVD in adult life. This process is known as Developmental Origins of Health and Disease (DOHaD) or fetal programming of CVD. The influence of fetal life for the future cardiovascular health of the individual has been evidenced by numerous epidemiologic studies in populations suffering from starvation during intrauterine life. Furthermore, experimental animal models have provided support and enabled exploring the underlying mechanisms. Oxidative stress seems to play a central role in fetal programming of CVD, both in the response of the feto-placental unit to the suboptimal intrauterine environment and in the alterations of physiologic systems of cardiovascular control, ultimately leading to disease. This review aims to summarize current knowledge on the alterations in oxidative balance in response to fetal stress factors covering two aspects. Firstly, the evidence from human studies of the implication of oxidative stress in LBW induced by suboptimal conditions during intrauterine life, emphasizing the role of the placenta. In the second part we summarize data on specific redox alterations in key cardiovascular control organs induced by exposure to known stress factors in experimental animals and discuss the emerging role of the mitochondria.
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Physiological stress response is a crucial adaptive mechanism for prey species survival. This paper aims to identify the main environmental and/or individual factors better explaining the stress response in Wood mice, Apodemus sylvaticus. We analyzed alterations in fecal glucocorticoid metabolite (FCM) concentration - extensively used as an accurate measure of the physiological stress response - of wild mice fecal samples seasonally collected during three years. Then, support vector machines were built to predict said concentration according to different stressors. These statistical tools appear to be particularly suitable for small datasets with substantial number of dimensions, corroborating that the stress response is an extremely complex process in which multiple factors can simultaneously partake in a context-dependent manner, i.e., the role of each potential stressor varies in time depending on other stressors. However, air-humidity, temperature and body-weight allowed us to explain the FCM fluctuation in 98% of our samples. The relevance of air-humidity and temperature altering FCM level could be linked to the presence of an abundant vegetation cover and, therefore, to food availability and predation risk perception. Body-weight might be related to the stress produced by reproduction and other intraspecific relationships such as social dominance or territorial behavior.
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Fezes , Glucocorticoides/metabolismo , Murinae/fisiologia , Estresse Fisiológico , Máquina de Vetores de Suporte , Animais , Comportamento Animal/fisiologia , Peso Corporal/fisiologia , CamundongosRESUMO
BACKGROUND AND AIMS: Fetal undernutrition is a risk factor for heart disease in both genders, despite the protection of women against hypertension development. Using a rat model of maternal undernutrition (MUN) we aimed to assess possible sex differences in the development of cardiac alterations and the implication of hypertension and cardiac oxidative stress. METHODS: Male and female offspring from rats fed ad libitum (control) or with 50% of the normal daily intake during the second half of gestation (MUN) were used. Heart weight/body weight ratio (HW/BW), hemodynamic parameters (anaesthetized rats) and plasma brain natriuretic peptide (BNP, ELISA) were assessed in 21-day, 6-month and 22-month old rats. Plasma testosterone (ELISA) and cardiac protein expression of enzymes related to reactive oxygen species synthesis (p22phox, xanthine-oxidase) and degradation (catalase, Cu/Zn-SOD, Mn-SOD, Ec-SOD) were evaluated in 21-day and 6-month old rats (Western Blot). Heart structure and function was studied at the age of 22 months (echocardiography). RESULTS: At the age of 21 days MUN males exhibited significantly larger HW/BW and cardiac p22phox expression while females had reduced p22phox expression, compared to their respective sex-matched controls. At the age of 6-months, MUN males showed significantly larger blood pressure and cardiac xanthine-oxidase expression; MUN females were normotensive and had a lower cardiac expression of antioxidant enzymes, compared to their respective sex-matched controls. At the age of 22 months, both MUN males and females showed larger HW/BW and left ventricular mass and lower ejection fraction compared to sex-matched controls; only MUN males exhibited hypertension and a larger plasma BNP compared to aged male controls. CONCLUSIONS: 1) During perinatal life females exposed to fetal undernutrition are protected from cardiac alterations, but in ageing they exhibit ventricular hypertrophy and functional loss, like MUN males; 2) cardiac oxidative stress might be implicated in the observed heart alterations in both sexes and 3) the severity of cardiac damage might be greater in males due to hypertension.
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Transtornos da Nutrição Fetal , Hipertensão/metabolismo , Estresse Oxidativo , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Animais , Peso Corporal , Feminino , Hemodinâmica , Hormônios/sangue , Hipertensão/sangue , Hipertensão/patologia , Hipertensão/fisiopatologia , Masculino , Mães , Miocárdio/metabolismo , Miocárdio/patologia , Peptídeo Natriurético Encefálico/metabolismo , Tamanho do Órgão , Gravidez , Efeitos Tardios da Exposição Pré-Natal/sangue , Efeitos Tardios da Exposição Pré-Natal/patologia , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Fatores de Risco , Caracteres Sexuais , Fatores de TempoRESUMO
INTRODUCTION: Oxidative stress is present in pregnancy complications. However, it is unknown if early maternal antioxidant status could influence later development of complications. The use of assisted reproduction techniques (ART) is rising due to the delay of first pregnancy and there is scarce information on its influence on oxidative balance. OBJECTIVE: To assess the possible relationship between maternal plasma antioxidant status in first trimester of gestation with later development of pregnancy complications, evaluating the influence of ART and nutrition. METHODS: Plasma from 98 healthy pregnant women was obtained at week 10, nutrition questionnaires filled and women were followed until delivery. We evaluated biomarkers of oxidative damage (carbonyls, malondialdehyde-MDA), antioxidants (thiols, reduced glutathione, phenolic compounds, catalase and superoxide dismutase activities) by spectrophotometry/fluorimetry and melatonin (ELISA). Antioxidant status score (Antiox-S) was calculated as the computation of antioxidants. Diet-antioxidants relationship was evaluated through multiple correspondence analysis. RESULTS: Melatonin and carbonyls exhibited a negative correlation. No difference in oxidative damage was found between groups, but Antiox-S was significantly lower in women who developed complications. No differences in oxidative damage or Antiox-S were found between ART and no-ART pregnancies. High consumption of foods of vegetable origin cluster with high plasma levels of phenolic compounds and with high Antiox-S. CONCLUSIONS: In early normal gestation, low plasma antioxidant status, assessed through a global score, associates with later development of pregnancy complications. Larger population studies could help to determine the value of Antiox-S as predictive tool and the relevance of nutrition on maternal antioxidant status.
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Estresse Oxidativo/fisiologia , Complicações na Gravidez/sangue , Primeiro Trimestre da Gravidez/sangue , Adulto , Biomarcadores/sangue , Catalase/metabolismo , Dieta , Feminino , Glutationa/sangue , Glutationa Peroxidase/metabolismo , Humanos , Malondialdeído/sangue , Melatonina/sangue , Gravidez , Superóxido Dismutase/metabolismoRESUMO
The aim of this study was to characterize alterations in vascular structure and mechanics in murine mesenteric arteries from obese non-hypertensive mice, as well as their relationship with adipokines. Four-week old C57BL/6J male mice were assigned either to a control (C, 10% kcal from fat) or a high-fat diet (HFD, 45% kcal from fat) for 32weeks. HFD animals weighed 30% more than controls (p<0.001), exhibited similar blood pressure, increased leptin, insulin and superoxide anion (O2(-)) levels, and reduced adiponectin levels and nitric oxide (NO) bioavailability. Arterial structure showed an outward remodeling with an increase in total number of both adventitial and smooth muscle cells in HFD. Moreover, HFD mice exhibited an increased arterial stiffness assessed by ß-values (C=2.4±0.5 vs HFD=5.3±0.8; p<0.05) and aortic pulse wave velocity (PWV, C=3.4±0.1 vs HFD=3.9±0.1; p<0.05). ß-Values and PWV positively correlated with leptin, insulin or O2(-) levels, whereas they negatively correlated with adiponectin levels and NO bioavailability (p<0.01). A reduction in fenestrae number together with an increase in type-I collagen amount (p<0.05) were observed in HFD. These data demonstrate that HFD accounts for the development of vascular remodeling and arterial stiffness associated with adipokine dysregulation and oxidative stress, independently of hypertension development.
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Adipocinas/sangue , Artérias Mesentéricas/fisiopatologia , Obesidade/imunologia , Rigidez Vascular/fisiologia , Adipocinas/imunologia , Animais , Dieta Hiperlipídica/efeitos adversos , Endotélio Vascular/imunologia , Masculino , Artérias Mesentéricas/imunologia , Artérias Mesentéricas/metabolismo , Artérias Mesentéricas/patologia , Camundongos Endogâmicos C57BL , Microscopia Confocal , Obesidade/metabolismo , Obesidade/patologia , Obesidade/fisiopatologia , Estresse Oxidativo/imunologia , Análise de Onda de Pulso , Remodelação Vascular/fisiologia , Rigidez Vascular/imunologiaRESUMO
Physical features, bioactive compounds and total antioxidant capacity (TAC) of coloured quinoa varieties (Chenopodium quinoa Willd.) from Peruvian Altiplano were studied. Quinoa seeds did not show a pure red colour, but a mixture which corresponded to different fractal colour values (51.0-71.8), and they varied from small to large size. Regarding bioactive compounds, total phenolic (1.23-3.24mg gallic acid equivalents/g) and flavonol contents (0.47-2.55mg quercetin equivalents/g) were highly correlated (r=0.910). Betalains content (0.15-6.10mg/100g) was correlated with L colour parameter (r=-0.569), total phenolics (r=0.703) and flavonols content (r=0.718). Ratio of betaxanthins to betacyanins (0.0-1.41) was negatively correlated with L value (r=-0.744). Whereas, high TAC values (119.8-335.9mmol Trolox equivalents/kg) were negatively correlated with L value (r=-0.779), but positively with betalains (r=0.730), as well as with free (r=0.639), bound (r=0.558) and total phenolic compounds (r=0.676). Unexploited coloured quinoa seeds are proposed as a valuable natural source of phenolics and betalains with high antioxidant capacity.
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Betalaínas/química , Chenopodium quinoa/química , Hidroxibenzoatos/química , Fenóis/análise , Sementes/química , Antioxidantes , PeruRESUMO
Long-term chronic intermittent exposure to altitude hypoxia is a labor phenomenon requiring further research. Using a rat model, we examined whether this type of exposure differed from chronic exposure in terms of pulmonary artery remodeling and other features. Rats were subjected to chronic hypoxia (CH, n = 9) and long-term intermittent hypoxia (CIH2x2; 2 days of hypoxia/2 days of normoxia, n = 10) in a chamber (428 Torr, 4,600 m of altitude) for 46 days and compared to rats under normoxia (NX, n = 10). Body weight, hematocrit, and right ventricle ratio were measured. Pulmonary artery remodeling was assessed using confocal microscopy of tissues stained with a nuclear dye (DAPI) and CD11b antibody. Both hypoxic conditions exhibited increased hematocrit and hypertrophy of the right ventricle, tunica adventitia, and tunica media, with no changes in lumen size. The medial hypertrophy area (larger in CH) depicted a significant increase in smooth muscle cell number. Additionally, CIH2x2 increased the adventitial hypertrophy area, with an increased cellularity and a larger prevalence of clustered inflammatory cells. In conclusion, CIH2x2 elicits milder effects on pulmonary artery medial layer muscularization and subsequent right ventricular hypertrophy than CH. However, CIH2x2 induces greater and characteristic alterations of the adventitial layer.
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Túnica Adventícia , Doença da Altitude , Hipóxia , Artéria Pulmonar , Remodelação Vascular , Túnica Adventícia/patologia , Túnica Adventícia/fisiopatologia , Doença da Altitude/patologia , Doença da Altitude/fisiopatologia , Animais , Modelos Animais de Doenças , Hipóxia/patologia , Hipóxia/fisiopatologia , Artéria Pulmonar/patologia , Artéria Pulmonar/fisiopatologia , Ratos , Ratos WistarRESUMO
BACKGROUND: The hypothesis of this study is that long-term high-fat diets (HFD) induce perivascular adipose tissue (PVAT) dysfunction characterized by a redox imbalance, which might contribute to aggravate endothelial dysfunction in obesity. METHODS AND RESULTS: C57BL/6J mice were fed either control or HFD (45% kcal from fat) for 32 weeks. Body weight, lumbar and mesenteric adipose tissue weights were significantly higher in HFD animals compared to controls. The anticontractile effect of PVAT in mesenteric arteries (MA) was lost after 32 week HFD and mesenteric endothelial-dependent relaxation was significantly impaired in presence of PVAT in HFD mice (Emaxâ=â71.0±5.1 vs Emaxâ=â58.5±4.2, p<0.001). The inhibitory effect of L-NAME on Ach-induced relaxation was less intense in the HFD group compared with controls suggesting a reduction of endothelial NO availability. Expression of eNOS and NO bioavailability were reduced in MA and almost undetectable in mesenteric PVAT of the HFD group. Superoxide levels and NOX activity were higher in PVAT of HFD mice. Apocynin only reduced contractile responses to NA in HFD animals. Expression of ec-SOD and total SOD activity were significantly reduced in PVAT of HFD mice. No changes were observed in Mn-SOD, Cu/Zn-SOD or catalase. The ratio [GSSG]/([GSH]+[GSSG]) was 2-fold higher in the mesenteric PVAT from HFD animals compared to controls. CONCLUSIONS: We suggest that the imbalance between pro-oxidant (NOX, superoxide anions, hydrogen peroxide) and anti-oxidant (eNOS, NO, ecSOD, GSSG) mechanisms in PVAT after long-term HFD might contribute to the aggravation of endothelial dysfunction.
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Tecido Adiposo/metabolismo , Dieta Hiperlipídica/efeitos adversos , Animais , Peso Corporal/fisiologia , Peróxido de Hidrogênio/metabolismo , Masculino , Artérias Mesentéricas/metabolismo , Artérias Mesentéricas/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Oxirredução , Superóxidos/metabolismoRESUMO
Work at high altitude in shifts exposes humans to a new form of chronic intermittent hypoxia, with still unknown health consequences. We have established a rat model resembling this situation, which develops a milder form of right ventricular hypertrophy and pulmonary artery remodelling compared to continuous chronic exposure. We aimed to compare the alterations in pulmonary artery nitric oxide (NO) availability induced by these forms of hypoxia and the mechanisms implicated. Rats were exposed for 46 days to normoxia or hypobaric hypoxia, either continuous (CH) or intermittent (2 day shifts, CIH2x2), and assessed: NO and superoxide anion availability (fluorescent indicators and confocal microscopy); expression of phosphorylated endothelial NO synthase (eNOS), NADPH-oxidase (p22phox), and 3-nitrotyrosine (western blotting); and NADPH-oxidase location (immunohistochemistry). Compared to normoxia, (1) NO availability was reduced and superoxide anion was increased in both hypoxic groups, with a larger effect in CH, (2) eNOS expression was only reduced in CH, (3) NADPH-oxidase was similarly increased in both hypoxic groups, and (4) 3-nitrotyrosine was increased to a larger extent in CH. In conclusion, intermittent hypoxia reduces NO availability through superoxide anion destruction, without reducing its synthesis, while continuous hypoxia affects both, producing larger nitrosative damage which could be related to the more severe cardiovascular alterations.
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Hipertrofia Ventricular Direita/metabolismo , Hipóxia/metabolismo , Óxido Nítrico/metabolismo , Superóxidos/metabolismo , Animais , Hipertrofia Ventricular Direita/patologia , Hipóxia/patologia , Masculino , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidase 1 , Óxido Nítrico Sintase Tipo III/metabolismo , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Ratos , Ratos WistarRESUMO
Dietary treatment with high-fat diets (HFD) triggers diabetes and hyperleptinemia, concomitantly with a partial state of leptin resistance that affects hepatic and adipose tissue but not the heart. In this context, characterized by widespread steatosis, cardiac lipid content remains unchanged. As previously reported, HFD-evoked hyperleptinemia could be a pivotal element contributing to increase fatty-acid (FA) metabolism in the heart and to prevent cardiac steatosis. This metabolic adaptation might theoretically reduce energy efficiency in cardiomyocytes and lead to cardiac electrophysiological remodeling. Therefore the aim of the current study has been to investigate the impact of long-term HFD on cardiac metabolism and electrophysiological properties of the principal ionic currents responsible of the action potential duration in mouse cardiomyocytes. Male C57BL/6J mice were fed a control (10 kcal% from fat) or HFD (45 kcal% from fat) during 32 weeks. Quantification of enzymatic activities regulating mitochondrial uptake of pyruvate and FA showed an increase of both carnitine-palmitoyltransferase and citrate synthase activities together with a decrease of lactate dehydrogenase and pyruvate dehydrogenase activities. Increased expression of uncoupling protein-3, Mn-, and Cu/Zn-superoxide dismutases and catalase were also detected. Total glutathione/oxidized glutathione ratios were unaffected by HFD. These data suggest that HFD triggers adaptive mechanisms aimed at (i) facilitating FA catabolism, and (ii) preventing oxidative stress. All these changes did not affect the duration of action potentials in cardiomyocytes and only slightly modified electrocardiographic parameters.
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The assessment of collagen content in tissues is important in biomedical research, since this protein is altered in numerous diseases. Hydroxyproline and Sirius red based assays are the most common methods for collagen quantification. However, these procedures have some pitfalls, such as the requirement of oxygen-free medium or expensive equipment and large sample size or being unsuitable for hydrolyzed collagen, respectively. Our objective was to develop a specific, versatile, and user-friendly quantitative method applicable to small tissue samples and extracts obtained from elastin purification, therefore, suitable for simultaneous quantification of elastin. This method is based on the binding of Sirius red to collagen present in a sample immobilized on a PVDF membrane, as in the dot-blot technique, and quantified by a scanner and image analysis software. Sample loading, Sirius red concentration, temperature and incubation time, type of standard substance, albumin interference, and quantification time are optimized. The method enabled the quantification of (1) intact collagen in several rat tissue homogenates, including small resistance-sized arteries, (2) partially hydrolyzed collagen obtained from NaOH extracts, compatible with elastin purification, and (3) for the detection of differences in collagen content between hypertensive and normotensive rats. We conclude that the developed technique can be widely used since it is versatile (quantifies intact and hydrolyzed collagen), requires small sample volumes, is user-friendly (low-cost, easy to use, minimum toxic materials, and reduced time of test), and is specific (minimal interference with serum albumin).
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Compostos Azo , Colágeno/imunologia , Immunoblotting/métodos , Animais , Masculino , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Distribuição TecidualRESUMO
A new microplate-adapted DPPH rapid assay was developed to assess the antioxidant capacity of pure compounds and foods. The assay was carried out in buffered medium (methanol: 10mmol/l Tris buffer pH 7.5, 1:1 v/v) and reaction was completed at 10min. The scavenging capacity index (SCI), a theoretical antioxidant parameter directly related to the antioxidant capacity of samples, was calculated. SCI for pure compounds: gallic acid (6.76±0.08), quercetin (7.89±0.24), catechin (6.05±0.23), trolox (2.32±0.03), ascorbic acid (2.52±0.15) and gluthatione (1.08±0.08) and foods (µmol DPPH scavenged/100ml): tropical juice (655.62±12.18), mediterraneo juice (702.87±11.13), apple juice (212.52±17.22), pomegranate juice (319.83±9.45), red grape nectar (1093.05±18.69), Don Simon orange juice (632.94±17.22) and date syrup (15992.34±250.7) were comparable to those in previous reports using the classic DPPH assay. The relative standard deviation (RSD) for the SCI on the same and different days was less than 8.12% in all cases.
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Bebidas/análise , Sequestradores de Radicais Livres/química , Frutas/química , Ensaios de Triagem em Larga Escala/métodos , Malus/química , Preparações de Plantas/química , Vitis/química , Compostos de Bifenilo/química , Ensaios de Triagem em Larga Escala/instrumentação , Picratos/químicaRESUMO
BACKGROUND: Chronic venous insufficiency (CVI) represents a social and health care problem because it affects working age populations, particularly in jobs requiring orthostasis, has no effective pharmacologic treatment, and requires surgery. Oxidative stress is present in varicose veins, but whether this is reflected in the plasma is controversial. We aimed to quantify plasma oxidative stress biomarkers in the early stages of CVI and calculate a global index of oxidative stress representative of the disease. METHODS: Plasma was obtained from blood samples of nine patients with CEAP C2 stage CVI and 10 healthy controls. Biomarkers related to antioxidant defense systems (total thiols, reduced glutathione, uric acid, total antioxidant capacity, catalase), oxidative damage (malondialdehyde-bound protein, protein carbonyls, advanced oxidation products, and 3-nitrotyrosine), and activity of enzymes producing key free radicals (xanthine oxidase and myeloperoxidase) were assessed. RESULTS: Compared with the controls, CVI patients exhibited decreased catalase activity and thiol levels and increased malondialdehyde-bound protein and protein carbonyls. These parameters were used to calculate the global index of oxidative stress in CVI, which was significantly different between groups. CONCLUSIONS: It is possible to detect significant changes in plasma oxidative stress biomarkers in early stages of CVI and to calculate a global index representative of the oxidative status in an individual. This index, with the appropriate validation in a larger population, could be used for early detection or progression of CVI.
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Estresse Oxidativo , Veias/metabolismo , Insuficiência Venosa/sangue , Adulto , Produtos da Oxidação Avançada de Proteínas/sangue , Biomarcadores/sangue , Estudos de Casos e Controles , Catalase/sangue , Doença Crônica , Progressão da Doença , Diagnóstico Precoce , Feminino , Glutationa/sangue , Humanos , Masculino , Malondialdeído/sangue , Pessoa de Meia-Idade , Peroxidase/sangue , Valor Preditivo dos Testes , Carbonilação Proteica , Compostos de Sulfidrila/sangue , Tirosina/análogos & derivados , Tirosina/sangue , Ultrassonografia Doppler em Cores , Ácido Úrico/sangue , Veias/diagnóstico por imagem , Insuficiência Venosa/diagnóstico por imagem , Xantina Oxidase/sangueRESUMO
Leptin causes vasodilatation both by endothelium-dependent and -independent mechanisms. Leptin is synthesized by perivascular adipose tissue (PVAT). The hypothesis of this study is that a decrease of leptin production in PVAT of spontaneously hypertensive rats (SHR) might contribute to a diminished paracrine anticontractile effect of the hormone. We have determined in aorta from Wistar-Kyoto (WKY) and SHR (i) leptin mRNA and protein levels in PVAT, (ii) the effect of leptin and PVAT on contractile responses, and (iii) leptin-induced relaxation and nitric oxide (NO) production. Leptin mRNA and protein expression were significantly lower in PVAT from SHR. Concentration-response curves to angiotensin II were significantly blunted in presence of PVAT as well as by exogenous leptin (10(-9) M) only in WKY. This anticontractile effect was endothelium-dependent. Vasodilatation induced by leptin was smaller in SHR than in WKY, and was also endothelium-dependent. Moreover, release of endothelial NO in response to acute leptin was higher in WKY compared to SHR, but completely abolished in the absence of endothelium. In conclusion, the reduced anticontractile effect of PVAT in SHR might be attributed to a reduced PVAT-derived leptin and to an abrogated effect of leptin on endothelial NO release probably due to an impaired activation of endothelial NO synthase.
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BACKGROUND: Liver growth factor (LGF) is an albumin-bilirubin complex with antioxidant actions in vitro. In spontaneously hypertensive rats (SHRs), short LGF treatment exerts antihypertensive and antifibrotic effects. METHOD: We aimed to determine if LGF treatment (4 i.p. injections, 4.5âµg/rat over 12 days) reduces oxidative stress in SHRs using Wistar-Kyoto (WKY) as control strain. We assessed the following: plasma oxidative stress biomarkers [protein-bound malondialdehyde (MDA); protein carbonyls and advanced glycation end products (AGEs)]; superoxide anion basal production in carotid artery-derived vascular smooth muscle cells (VSMCs) detected by dihydroethidium and confocal microscopy; and expression (western blot) and activities (spectroscopic methods) of NADPH and xanthine oxidases, CuZn, Mn and extracellular superoxide dismutases (SODs) and catalase in carotid arteries. RESULTS: LGF treatment had the following effects: reversed the increase in plasma MDA and protein carbonyls and VSMC superoxide anion levels observed in SHRs, without any effect on WKY strain; reversed the alterations in SHR vascular p22phox expression as well as NADPH oxidase, xanthine oxidase and catalase activities; had no effect on vascular CuZn-SOD and Mn-SOD expression or total SOD activity; and reversed the elevation in SHR vascular glycated/free extracellular-SOD expression ratio and plasma glucose without changes in plasma AGEs. CONCLUSION: LGF treatment of SHRs normalizes the level of plasma oxidative stress biomarkers through a reduction of vascular superoxide anion produced by NADPH and xanthine oxidases. These effects might be linked to the cardiovascular regenerative actions of LGF.