RESUMO
The aim of this study was to determine the apoptotic pathways and mechanisms involved in electronegative LDL [LDL(-)]-induced apoptosis in RAW 264.7 macrophages and the role of Nrf2 in this process. Incubation of RAW 264.7 macrophages with LDL(-) for 24 h resulted in dose-dependent cell death. Activated caspases were shown to be involved in the apoptosis induced by LDL(-); incubation with the broad caspase inhibitor z-VAD prevented apoptosis in LDL(-)-treated cells. CD95 (Fas), CD95 ligand (FasL), CD36 and the tumor necrosis factor (TNF) ligand Tnfsf10 were overexpressed in LDL(-)-treated cells. However, Bax, Bcl-2 and Mcl-1 protein levels remained unchanged after LDL(-) treatment. LDL(-) promoted hyperpolarization of the mitochondrial membrane, elevated reactive oxygen species (ROS) production and translocation of Nrf2 to the nucleus, a process absent in cells treated with native LDL. Elicited peritoneal macrophages from Nrf2-deficient mice exhibited an elevated apoptotic response after challenge with LDL(-), together with an increase in the production of ROS in the absence of alterations in CD36 expression. These results provide evidence that CD36 expression induced by LDL(-) is Nrf2-dependent. Also, it was demonstrated that Nrf2 acts as a compensatory mechanism of LDL(-)-induced apoptosis in macrophages.
Assuntos
Apoptose , Lipoproteínas LDL/metabolismo , Macrófagos Peritoneais/patologia , Fator 2 Relacionado a NF-E2/fisiologia , Animais , Biomarcadores/metabolismo , Western Blotting , Antígenos CD36/genética , Antígenos CD36/metabolismo , Proteína Ligante Fas/genética , Proteína Ligante Fas/metabolismo , Feminino , Imunofluorescência , Perfilação da Expressão Gênica , Humanos , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Oxidative modification of low-density lipoprotein (LDL) has been demonstrated in patients with end-stage renal disease, where it is associated with oxidative stress and plays a key role in the pathogenesis of atherosclerosis. In this context, the generation of minimally oxidized LDL, also called electronegative LDL [LDL(-)], has been associated with active disease, and is a detectable sign of atherogenic tendencies. The purpose of this study was to evaluate serum LDL(-) levels and anti-LDL(-) IgG autoantibodies in end-stage renal disease patients on dialysis, comparing patients on hemodialysis (HD), peritoneal dialysis (PD) and a control group. In addition, the serum lipid profile, nutritional status, biochemical data and parameters of mineral metabolism were also evaluated. METHODS: The serum levels of LDL(-) and anti-LDL(-) IgG autoantibodies were measured in 25 patients undergoing HD and 11 patients undergoing PD at the Centro Integrado de Nefrologia, Rio de Janeiro, Brazil. Ten healthy subjects served as a control group. Serum levels of albumin, total cholesterol, triglycerides and lipoproteins were measured. Calculations of subjects' body mass index and measurements of waist circumference, triceps skin fold and arm muscle area were performed. Measurements of hematocrit, serum blood urea nitrogen, creatinine, parathyroid hormone, phosphorus and calcium were taken. RESULTS: Levels of LDL(-) were higher in HD patients (575.6 +/- 233.1 microg/ml) as compared to PD patients (223.4 +/- 117.5 microg/ml, p < 0.05), which in turn were higher than in the control group (54.9 +/- 33.3 mug/ml, p < 0.01). The anti-LDL(-) IgG autoantibodies were increased in controls (0.36 +/- 0.09 microg/ml) as compared to PD (0.28 +/- 0.12 microg/ml, p < 0.001) and HD patients (0.2 +/- 0.1 microg/ml, p < 0.001). The mean values of total cholesterol and LDL were considered high in the PD group, whereas the mean triceps skin fold was significantly lower in the HD group. CONCLUSION: Levels of LDL(-) are higher in renal patients on dialysis than in normal individuals, and are reciprocally related to IgG autoantibodies. LDL(-) may be a useful marker of oxidative stress, and this study suggests that HD patients are more susceptible to cardiovascular risk due to this condition. Moreover, autoantibodies reactive to LDL(-) may have protective effects in chronic kidney disease.
Assuntos
Autoanticorpos/sangue , Imunoglobulina G/sangue , Falência Renal Crônica/sangue , Falência Renal Crônica/terapia , Lipoproteínas LDL/sangue , Diálise Peritoneal , Diálise Renal , Adulto , Análise de Variância , Biomarcadores/sangue , Pesos e Medidas Corporais , Colesterol/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Falência Renal Crônica/imunologia , Lipoproteínas LDL/imunologia , Lipoproteínas LDL/metabolismo , Masculino , Pessoa de Meia-Idade , Estado Nutricional , Estatísticas não ParamétricasRESUMO
The conversion of oxyhemoglobin to methemoglobin has been shown via spectrophotometric, circular dichroism and polarographic studies to be accelerated by delta-aminolevulinic acid, a major heme-precursor accumulated in a number of heme-linked pathologies. Concomitantly, delta-aminolevulinic acid undergoes aerobic oxidation. The intermediacy of oxygen radicals in these processes was evidenced by the inhibitory effect of catalase, superoxide dismutase and mannitol. These results are relevant to the exacerbated production of active oxygen species in intermittent acute porphyria and saturnism carriers.
Assuntos
Ácido Aminolevulínico/metabolismo , Ácidos Levulínicos/metabolismo , Oxigênio/metabolismo , Oxiemoglobinas/metabolismo , Animais , Catalase/metabolismo , Bovinos , Humanos , Cinética , Manitol/metabolismo , Metemoglobina/metabolismo , Oxirredução , Consumo de Oxigênio , Porfirias/metabolismo , Ovinos , Superóxido Dismutase/metabolismoRESUMO
The oxidative burst of polymorphonuclear neutrophils (PMN) and their ability to inhibit Paracoccidioides brasiliensis growth was studied in susceptible (B10.A) and resistant (A/J) mice. The cells were obtained after subcutaneous inoculation in air pouches, yielding highly pure PMN preparations; the number of cells was similar for both strains at 24 h and five times higher in the resistant strain at 15 days. The oxidative metabolism of these PMN was evaluated by the luminol and lucigen-enhanced chemiluminescence upon stimulation with PMA or killed P. brasiliensis (Pb). At 24 h of infection PMN from both strains showed similar responses. However, at 15 days a great enhancement of the Pb-stimulated luminol-enhanced chemiluminescence was observed only in PMN from resistant mice. Such increase was markedly inhibited by the addition of catalase. Independent of the mouse strain or time of infection of lucigen-enhanced chemiluminescence showed the same intensity. The lucigen-enhanced chemiluminescence of PMN without stimuli from resistant mice did not change with the time of infection, however, after 15 days of infection a significantly lower chemiluminescence was detected with PMN from susceptible mice. At 15 days of infection the PMN from B10.A were unable to kill P. brasiliensis yeast cells in vitro. Because the lucigenin- and luminol-enhanced chemiluminescence detects, respectively, the O2- production and the myeloperoxidase/hydrogen peroxide halide system, the present data show parallels between deficiency in the production of oxygen-reactive species by PMN and lower fungicidal activity.
Assuntos
Neutrófilos/imunologia , Neutrófilos/metabolismo , Paracoccidioides , Paracoccidioidomicose/imunologia , Explosão Respiratória , Acridinas , Animais , Suscetibilidade a Doenças , Medições Luminescentes , Luminol , Masculino , Camundongos , Camundongos Endogâmicos A , Neutrófilos/citologia , Paracoccidioidomicose/microbiologiaRESUMO
Angiogenesis is a pivotal process of homeostasis and tissue repair, but it also favours neovascularisation syndromes and cancer nutrition. The chemical mediation of angiogenesis is complex, involving a balance between serine proteases and their inhibitors. We addressed the mechanisms of action of a Kunitz serine protease inhibitor (KPI) on spontaneous angiogenesis, using Amblyomin-X, a KPI designed from the cDNA library of the Amblyomma cajennense tick. Amblyomin-X treatment (10-1000 ng/10 µL; each 48 h; 3 times) reduced the number of vessels in the subcutaneous dorsal tissue of male Swiss mice, as measured by intravital microscopy, haematoxylin-eosin staining, and PECAM-1 immunofluorescence labeling. Incubation of Amblyomin-X with t-End endothelial cells, a murine endothelial microvascular lineage, did not alter cell proliferation, cell-cycle phases, necrosis and apoptosis, and the production of nitric oxide and prostaglandin E2. Nevertheless, Amblyomin-X treatment reduced t-End migration and adhesion to Matrigel(®), and inhibited the VEGF-A secretion and VCAM-1 and ß3 integrin expressions by posttranscriptional pathways. Together, data herein outline novel posttranscriptional mechanisms of KPIs on endothelial cells during angiogenesis and point out the possible application of Amblyomin-X as a local inhibitor to undesired neovascularisation process.
Assuntos
Adesão Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas e Peptídeos Salivares/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas de Artrópodes , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dinoprostona/metabolismo , Células Endoteliais/metabolismo , Biblioteca Gênica , Masculino , Camundongos , Neovascularização Fisiológica/efeitos dos fármacos , Óxido Nítrico/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas e Peptídeos Salivares/genética , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Oxidative modification of LDL is evidenced by alterations in both the protein and lipid components of the particle. Progressive oxidation of the apoprotein is associated with loss of specific amino acids and a gradual increase in electronegativity. Electronegative LDL has been isolated from human plasma (LDL-) by several groups using liquid chromatographic techniques and appears to be oxidized based on increased lipid peroxide levels and cholesterol oxidation products (ChOx). Formation of LDL- also takes place following Cu(2+)-induced oxidation. Cu(2+)-induced oxidation caused a small fraction of the normal unoxidized LDL (n-LDL) to convert to LDL-during the oxidative lag phase while minimal increases in conjugated dienes were apparent. After the lag phase, there was a further increase in LDL-, a rapid accumulation of conjugated dienes, and another more electronegative particle was formed (LDL2-). By the end of the lag phase, approximately 30% and 12% of the total LDL converted to LDL- and LDL2-, respectively. Nearly 40% of the total ChOx formed was present by the end of the lag period, accompanied by small increases in conjugated dienes. The major products accumulating during this time were 7-ketocholesterol, cholesterol-beta-epoxide and 7-alpha-hydroxycholesterol. Accumulation of predominated during the subsequent propagation phase. At the end of propagation phase there was a six fold increase in conjugated dienes and total ChOx increased eight-fold. It appears that a subpopulation of LDL rapidly converts to LDL-, representing a mildly oxidized but oxidant sensitive LDL population. Oxidation of cholesterol accompanies these early events in LDL oxidation with formation of specific ChOx.
Assuntos
Colesterol/metabolismo , Lipoproteínas LDL/metabolismo , Adulto , Arteriosclerose/etiologia , Arteriosclerose/metabolismo , Colesterol/química , Cobre/metabolismo , Radicais Livres/metabolismo , Humanos , Técnicas In Vitro , Cinética , Peroxidação de Lipídeos , Lipoproteínas LDL/química , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Vitamina E/metabolismoRESUMO
Nitric oxide (NO) reacts with thiol-containing biomolecules to form S-nitrosothiols (RSNOs). RSNOs are considered as NO reservoirs as they generate NO by homolytic cleavage. Ceruloplasmin has recently been suggested to have a potent catalytic activity towards RSNO production. Considering that NO activity is impaired in hypercholesterolemia and that RSNOs may act as important NO donors, we investigated the relation between concentrations of ceruloplasmin and RSNOs in plasma of hypercholesterolemic (HC) patients compared to normolipidemic (N) controls. Concentrations of ceruloplasmin (0.36 +/- 0.07 x 0.49 +/- 0.11 mg/dl, N x HC), nitrate (19.10 +/- 12.03 x 40.19 +/- 18.70 microM, N x HC), RSNOs (0.25 +/- 0.20 x 0.54 +/- 0.26 microM, N x HC), nitrated LDL (19.51 +/- 6.98 x 35.29 +/- 17.57 nM nitro-BSA equivalents, N x HC), and cholesteryl ester-derived hydroxy/hydroperoxides (CEOOH, 0.19 +/- 0.06 x 1.46 +/- 0.97 microM) were increased in plasma of HC as compared to N. No difference was found for nitrite levels between the two groups (1.01 +/- 0.53 x 1.02 +/- 0.33 microM, N x HC). The concentrations of RSNOs, nitrate, and nitrated LDL were positively correlated to those of total cholesterol, LDL cholesterol, and apoB. Ceruloplasmin levels were directly correlated to apoB and apoE concentrations. Data suggest that: (i) ceruloplasmin may have a role in the enhancement of RSNOs found in hypercholesterolemia; (ii) the lower NO bioactivity associated with hypercholesterolemia is not related to a RSNOs paucity or a defective NO release from RSNOs; and (iii) the increased nitrotyrosine levels found in hypercholesterolemia indicate that superoxide radicals contribute to inactivation of NO, directly generated by NO synthase or originated by RSNO decomposition.
Assuntos
Ceruloplasmina/metabolismo , Hipercolesterolemia/sangue , Mercaptoetanol , Compostos Nitrosos/sangue , S-Nitrosotióis , Tirosina/análogos & derivados , Apolipoproteínas B/sangue , Ácido Ascórbico/sangue , Catálise , Colesterol/sangue , Ésteres do Colesterol/sangue , LDL-Colesterol/sangue , Humanos , Peróxido de Hidrogênio/sangue , Lipoproteínas LDL/sangue , Nitratos/sangue , Nitritos/sangue , Tirosina/sangue , Ácido Úrico/sangue , Vitamina E/sangueRESUMO
A highly sensitive and simple method for the determination of cholesteryl ester hydroperoxides (ChE-OOH) was developed using high-performance liquid chromatography (HPLC) with coulometric electrochemical detection. The lowest detectable level by this technique was 2 pmol for cholesteryl linoleate hydroperoxides at the signal-to-noise ratio of 3. This method was applied to the determination of ChE-OOH presumably present in human plasma. Although ChE-OOH could not be detected, the ChE-OOH level in the fluid was estimated to be less than 27 nM. It was found that the extraction efficiency of an internal standard, cholesteryl nervonate, was decreased by lowering its amount spiked to the plasma. The concentration of ChE-OOH in human plasma and plasma lipoprotein, which were peroxidized with a radical initiator in vitro, could be determined by use of this standard. HPLC-coulometric technique is, therefore, useful to measure the peroxidation of plasma lipids in vitro.
Assuntos
Ésteres do Colesterol/sangue , Peróxidos Lipídicos/sangue , Cromatografia Líquida de Alta Pressão/métodos , Eletroquímica/métodos , Humanos , Peroxidação de Lipídeos , Lipoproteínas/sangue , Lipoproteínas LDL/sangue , Lipoproteínas VLDL/sangue , Sensibilidade e EspecificidadeRESUMO
Free radicals superoxide (O(2)(-)) and nitric oxide (*NO) are generated by blood vessels and can rapidly react to produce a peroxynitrite anion (ONOO(-)), a powerful oxidant that modifies lipoproteins making them more atherogenic. The aim of this study was to investigate the effect of peroxynitrite-induced modifications on beta-very-low-density lipoprotein (beta-VLDL) as to its biodistribution and plasma clearance rate, as well as the uptake of these particles by THP-1 cells. After being injected into New Zealand White rabbits, the peroxynitrite-modified beta-VLDL (99mTc-per-beta-VLDL) was cleared from circulation faster than the native beta-VLDL (99mTc-nat-beta-VLDL) in both normocholesterolemic rabbits (NC) and in hypercholesterolemic rabbits (HC). In HC rabbits, the fractional clearance of 99mTc-labeled beta-VLDL was significantly lower than in NC rabbits. The in vivo studies showed that accumulation of 99mTc-labeled beta-VLDL, expressed per gram of tissue, followed the decreasing order: kidney > liver > spleen > adrenal gland >or= lung > aortic arch > heart >or= abdominal aorta > thoracic aorta > psoas muscle. The high accumulation in the kidneys suggests the processing of 99mTc-labeled apolipoproteins by receptors present in kidney cells. The accumulation of 99mTc-nat-beta-VLDL in the whole organ was the following: liver > kidney > heart > spleen > adrenal gland > aorta in HC and NC rabbits. The uptake of 99mTc-per-beta-VLDL by the spleen was greater than the uptake by the heart in both groups. The in vitro studies showed that the uptake of 99mTc-per-beta-VLDL by THP-1 cells was higher than that of 99mTc-nat-beta-VLDL. These results show that peroxynitrite-modified beta-VLDL is rapidly removed from plasma and accumulates in several tissues, mainly in the liver and kidney. This may be particularly important in hypercholesterolemic situations that could favor the accumulation of native and peroxynitrite-modified beta-VLDL in several tissues.
Assuntos
Lipoproteínas VLDL/farmacocinética , Animais , Masculino , Taxa de Depuração Metabólica , Ácido Peroxinitroso , Coelhos , Tecnécio/farmacocinética , Distribuição TecidualRESUMO
Low density lipoprotein (LDL) oxidation mediated by phorbol myristate acetate (PMA)- and formylmethionylleucylphenylalanine (FMLP) -stimulated human neutrophils was enhanced by 70% in the presence of ferritin. Iron released from ferritin by the superoxide anion generated in the respiratory burst of stimulated neutrophils is shown to be involved in lipoprotein oxidation. Ascorbate (100 microM), superoxide dismutase (10 micrograms/ml) and uric acid (430 microM) showed inhibitory effects of 30% [corrected], 70% and 50% on LDL oxidation, respectively. Ceruloplasmin (2.7 microM) potentiated LDL oxidation by stimulated neutrophils and ferritin, both alone and in the presence of methionine. Methionine (1 mM) and catalase (30 micrograms/ml) increased LDL oxidation by stimulated neutrophils and ferritin. These data suggest that LDL oxidation by stimulated neutrophils and ferritin may be relevant in inflammation when both neutrophils and ferritin are increased.
Assuntos
Ferritinas/farmacologia , Lipoproteínas LDL/metabolismo , Neutrófilos/metabolismo , Apoproteínas/farmacologia , Ácido Ascórbico/farmacologia , Catalase/farmacologia , Ceruloplasmina/farmacologia , Humanos , Peroxidação de Lipídeos , Metionina/farmacologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Oxirredução , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Substâncias Reativas com Ácido Tiobarbitúrico , Transferrina/farmacologia , Ácido Úrico/farmacologiaRESUMO
The metabolism of low density lipoprotein (LDL) oxidized with phorbol myristate acetate (PMA) stimulated neutrophils plus ferritin (LDLox) by human monocyte-derived macrophage (HMDM) was studied. Binding of 125I-labeled LDLox to HMDM and further uptake and degradation were higher than for native 125I-labeled LDL. LDLox seems to be taken up by HMDM through the scavenger receptor as indicated by competition studies with unlabeled native and autoxidized LDL. An increased concentration of cellular cholesteryl esters was observed in HMDM exposed to LDLox. Oxidative modification of LDL increased its electrophoretic migration on agarose gel and also the fragmentation of apolipoprotein B. Data suggest that LDLox is incorporated by human macrophages and can potentially induce foam-cell formation.
Assuntos
Ferritinas/farmacologia , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Neutrófilos/metabolismo , Ligação Competitiva , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Humanos , Masculino , OxirreduçãoRESUMO
An antioxidant defense system consisting of enzymes and non-enzymatic compounds prevents oxidative damage of lipoproteins in the plasma. When the activity of this system decreases or the reactive oxygen species (ROS) production increases, an oxidative stress may occur. Since fatty acids and triglyceride-rich emulsions can stimulate leukocytes to produce ROS, it is conceivable that raised plasma triglyceride-rich lipoproteins such as very low density lipoprotein (VLDL) may overload the antioxidant system. To test this hypothesis, we selected 14 patients with combined hyperlipidemia (HLP), in whom low density lipoprotein (LDL) and VLDL levels are elevated, as well as 18 hypercholesterolemic patients (HCH) with increased LDL levels and 19 controls (NL) to examine the trend for an imbalance between the production of oxidative species and the antioxidant defense system as challenged by increased plasma lipids. With this goal, plasma lipoprotein lipid fractions were determined and correlated with the release of ROS by leukocytes monitored by luminol-enhanced chemiluminescence. Plasma beta-carotene, alpha-tocopherol, lycopene and the lipoprotein lipid hydroperoxides were determined by high pressure liquid chromatography with electrochemical detection. HLP had lower plasma superoxide dismutase (SOD) activity (0.04 and 0.11 U/mg protein; P < 0.05) as well as lower concentrations of lycopene (0.1 and 0.2 nmol/mg cholesterol; P < 0.05) and beta-carotene (0.8 and 2.7 nmol/mg cholesterol; P < 0.05) in the plasma, as compared with NL. Moreover, HLP showed the highest ROS production by resting mononuclear leukocytes (MN) among the three study groups. When the results of the subjects of the three groups were taken together, the plasma triglyceride concentration was positively correlated to ROS release by resting polymorphonuclear leukocytes (PMN, r = 0.38, P = 0.04) and MN (r = 0.56, P < 0.005). Moreover, ROS release by resting MN was positively correlated with VLDL (r = 0.47, P = 0.02) and LDL (r = 0.57, P = 0.01) triglycerides. There was also a positive correlation between ROS release by stimulated PMN and VLDL (r = 0.44, P = 0.03) as well as LDL (r = 0.53, P = 0.01) triglycerides. High density lipoprotein (HDL) cholesterol showed a negative correlation with ROS release by resting MN (r = -0.48, P = 0.02) and resting PMN (r = -0.49, P = 0.01). VLDL susceptibility to copper (II) oxidation was not different among the three groups. Regarding LDL, there was an increased oxidizability in HLP group.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Hiperlipidemias/sangue , Leucócitos/metabolismo , Lipoproteínas/sangue , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Triglicerídeos/sangue , Adulto , Cromatografia Líquida de Alta Pressão , Feminino , Ferritinas/sangue , Humanos , Medições Luminescentes , Masculino , Pessoa de Meia-Idade , Superóxido Dismutase/sangueRESUMO
OBJECTIVES: Low density lipoprotein (LDL) does not constitute an homogenous fraction and it is known that the heavy LDL subfraction is potentially more atherogenic than the light one. Because concentration of LDL subfractions tend to be different in hyperlipidemias, it was verified whether these subfractions can also differ in sialic acid and neutral sugar content, as well as their resistance to oxidation. DESIGN AND METHODS: Two subfractions of low density lipoprotein (light LDL, density 1.019-1.034 g/mL and heavy LDL, density 1.034-1.063 g/mL) were isolated from the plasma of 17 patients with hypercholesterolemia, 11 with combined hyperlipidemia, 7 with hypertriglyceridemia, and 19 normolipidemic subjects. The content of sialic acids and neutral sugars of apo B was determined, respectively, by the periodate-thiobarbituric acid method and by reaction with phenol. The oxidation of LDL subfractions was determined by exposure to 5 microM copper (II) followed by the measurement of lipid hydroperoxides production by high performance liquid chromatography with electrochemical detection. RESULTS: The study groups did not differ in the neutral sugar content of LDL subfractions. However, compared to normolipidemic subjects, the sialic acid concentration of both LDL subfractions was lower in patients with hypercholesterolemia and hypertriglyceridemia and higher in those with combined hyperlipidemia (p < 0.05). In the hypercholesterolemia and combined hyperlipidemia groups, the lipid hydroperoxide content (microM) of heavy LDL was higher than in normolipidemic subjects (p < 0.05). CONCLUSIONS: The heavy LDL subfraction was more susceptible to oxidation in the patients with combined hyperlipidemia compared to controls and the other hyperlipidemic groups. The effect of sialic acids on heavy LDL oxidizability seems to vary according to the type of hyperlipidemia.
Assuntos
Hiperlipidemias/metabolismo , Lipoproteínas LDL/metabolismo , Ácidos Siálicos/sangue , Adulto , Antioxidantes/farmacologia , Estudos de Casos e Controles , Fracionamento Químico , Feminino , Humanos , Lipoproteínas LDL/química , Lipoproteínas LDL/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Ácido N-Acetilneuramínico , Oligossacarídeos/análise , Oligossacarídeos/química , Oxirredução , Ácidos Siálicos/química , Ácidos Siálicos/farmacologiaRESUMO
The relationship between free radical and scavenger enzymes has been found in the epileptic phenomena and reactive oxygen species have been implicated in seizure-induced neurodegeneration. Using the epilepsy model obtained by systemic administration of pilocarpine (PILO) in rats, we investigated the superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities as well as the hydroperoxide (HPx) concentration in the hippocampus of rats during status epilepticus (SE), silent and chronic periods. The enzyme activities as well as the HPx concentration were measured using spectrophotometric methods and the results compared to values obtained from saline-treated animals. The SOD activity decreased after long-lasting SE period and during the chronic phase. In addition, HPx levels increased in same periods whereas the GPx activity increased only in the hippocampus of animals submitted to 1 h of SE. Animals presenting partial seizures, those submitted to 5 h of SE and animals from the silent period (seizure free) showed normal levels of SOD, GPx and HPx. These results show a direct evidence of lipid peroxidation during seizure activity that could be responsible for neuronal damage in the hippocampus of rats, during the establishment of PILO model of epilepsy.
Assuntos
Epilepsia/metabolismo , Glutationa Peroxidase/metabolismo , Hipocampo/metabolismo , Peróxido de Hidrogênio/metabolismo , Superóxido Dismutase/metabolismo , Animais , Doença Crônica , Epilepsia/induzido quimicamente , Masculino , Agonistas Muscarínicos , Concentração Osmolar , Pilocarpina , Ratos , Ratos Wistar , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/metabolismoRESUMO
The present study examined the effect of diet supplementation of oxaloacetate precursors (aspartate and asparagine) and carnitine on muscle metabolism and exercise endurance. The results suggest that the diet supplementation increased the capacity of the muscle to utilize FFA and spare glycogen. Time to exhaustion was about 40% longer in the experimental group compared to the control, which received commercial diet only. These findings suggest that oxaloacetate may be important to determine the time to exhaustion during a prolonged and moderate exercise.
Assuntos
Asparagina/farmacologia , Ácido Aspártico/farmacologia , Carnitina/farmacologia , Músculo Esquelético/metabolismo , Esforço Físico/fisiologia , Animais , Asparagina/sangue , Ácido Aspártico/sangue , Glicemia/metabolismo , Carnitina/sangue , Citrato (si)-Sintase/metabolismo , Dieta , Ácidos Graxos/metabolismo , Glicogênio/metabolismo , Glicogênio Hepático/metabolismo , Masculino , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/enzimologia , Oxaloacetatos/metabolismo , Oxirredução , Condicionamento Físico Animal , Ratos , Ratos Wistar , NataçãoRESUMO
Oxidative processes leading to the generation of oxidized low density lipoprotein (oxLDL) particles have been suggested to be an important factor in the pathogenesis of atherosclerosis. After initiation of the oxidative process, LDL undergoes a progressive protein and lipid fragmentation. To understand this process and the role of oxLDL in various diseases of inflammatory origin, we have generated mouse monoclonal antibodies against copper-oxidized human LDL. Mice were immunized intrasplenically and after one intravenous boost the spleen cells were fused with the Sp2/0 hybridoma fusion partner. The hybridoma clones obtained after selection and cloning were analyzed for reactivity against oxLDL with various degrees of copper-mediated oxidative modifications. Three hybridoma clones were purified and further characterized. The following observations were made: 1) the intrasplenic route of immunization, avoiding the use of mycobacterial adjuvants, yielded a high frequency of positive clones; 2) the individual hybridomas reacted against LDL with various degrees of oxidative modifications; 3) the monoclonal antibodies could be used in ELISA and to detect oxLDL in immunohistochemical tissue staining, and 4) the monoclonal antibodies also detected oxLDL from hamsters and rabbits. We conclude that these monoclonal antibodies could be useful to further investigate the role of oxLDL in inflammation and in the immune response.
Assuntos
Anticorpos Monoclonais , Lipoproteínas LDL/imunologia , Estresse Oxidativo , Animais , CamundongosRESUMO
The biodistribution and removal from plasma (measured as fractional clearance rate, FCR, per hour) of native and oxidatively modified 99mtechnetium-labeled beta-very low density lipoprotein (99mTc-beta-VLDL) were investigated in hypercholesterolemic (HC) and control (C) three-month old New Zealand rabbits. The intracellular accumulation of beta-VLDL labeled with 99mTc was studied in vitro in THP-1 cells and monocyte-derived macrophages isolated from rabbits. After intravenous injection into C rabbits, copper-oxidized beta-VLDL (99mTc-ox-beta-VLDL) was cleared from the circulation faster (0.362 +/- 0.070/h) than native beta-VLDL (99mTc-nat-beta-VLDL, 0.241 +/- 0.070/h). In contrast, the FCR of 99mTc-ox-beta-VLDL in HC rabbits was lower (0.100 +/- 0.048/h) than that of 99mTc-nat-beta-VLDL (0.163 +/- 0.043/h). The hepatic uptake of radiolabeled lipoproteins was lower in HC rabbits (0.114 +/- 0.071% injected dose/g tissue for 99mTc-nat-beta-VLDL and 0.116 +/- 0.057% injected dose/g tissue for 99mTc-ox-beta-VLDL) than in C rabbits (0.301 +/- 0.113% injected dose/g tissue for 99mTc-nat-beta-VLDL and 0.305 +/- 0.149% injected dose/g tissue for 99mTc-ox-beta-VLDL). The uptake of 99mTc-nat-beta-VLDL and 99mTc-ox-beta-VLDL by atherosclerotic aorta lesions isolated from HC rabbits (99mTc-nat-beta-VLDL: 0.033 +/- 0.012% injected dose/g tissue and 99mTc-ox-beta-VLDL: 0.039 +/- 0.017% injected dose/g tissue) was higher in comparison to that of non-atherosclerotic aortas from C rabbits (99mTc-nat-beta-VLDL: 0.023 +/- 0.010% injected dose/g tissue and 99mTc-ox-beta-VLDL: 0.019 +/- 0.010% injected dose/g tissue). However, 99mTc-nat-beta-VLDL and 99mTc-ox-beta-VLDL were taken up by atherosclerotic lesions at similar rates. In vitro studies showed that both monocyte-derived macrophages isolated from rabbits and THP-1 macrophages significantly internalized more 99mTc-ox-beta-VLDL than 99mTc-nat-beta-VLDL. These results indicate that in cholesterol-fed rabbits 99mTc-ox-beta-VLDL is slowly cleared from plasma and accumulates in atherosclerotic lesions. However, although the extent of in vitro uptake of 99mTc-ox-beta-VLDL by macrophages was high, the in vivo accumulation of this radiolabeled lipoprotein by atherosclerotic lesions did not differ from that of 99mTc-nat-beta-VLDL.
Assuntos
Lipoproteínas VLDL/farmacocinética , Pertecnetato Tc 99m de Sódio/farmacocinética , Animais , Arteriosclerose/metabolismo , Lipídeos/sangue , Lipoproteínas VLDL/sangue , Lipoproteínas VLDL/isolamento & purificação , Ativação de Macrófagos/fisiologia , Masculino , Taxa de Depuração Metabólica , Coelhos , Pertecnetato Tc 99m de Sódio/sangue , Pertecnetato Tc 99m de Sódio/isolamento & purificação , Distribuição Tecidual/fisiologiaRESUMO
The objective of the present study was to identify disturbances of nitric oxide radical (.NO) metabolism and the formation of cholesterol oxidation products in human essential hypertension. The concentrations of.NO derivatives (nitrite, nitrate, S-nitrosothiols and nitrotyrosine), water and lipid-soluble antioxidants and cholesterol oxides were measured in plasma of 11 patients with mild essential hypertension (H: 57.8 +/- 9.7 years; blood pressure, 148.3 +/- 24.8/90.8 +/- 10.2 mmHg) and in 11 healthy subjects (N: 48.4 +/- 7.0 years; blood pressure, 119.4 +/- 9.4/75.0 +/- 8.0 mmHg). Nitrite, nitrate and S-nitrosothiols were measured by chemiluminescence and nitrotyrosine was determined by ELISA. Antioxidants were determined by reverse-phase HPLC and cholesterol oxides by gas chromatography. Hypertensive patients had reduced endothelium-dependent vasodilation in response to reactive hyperemia (H: 9.3 and N: 15.1% increase of diameter 90 s after hyperemia), and lower levels of ascorbate (H: 29.2 +/- 26.0, N: 54.2 +/- 24.9 micro M), urate (H: 108.5 +/- 18.9, N: 156.4 +/- 26.3 micro M), beta-carotene (H: 1.1 +/- 0.8, N: 2.5 +/- 1.2 nmol/mg cholesterol), and lycopene (H: 0.4 +/- 0.2, N: 0.7 +/- 0.2 nmol/mg cholesterol), in plasma, compared to normotensive subjects. The content of 7-ketocholesterol, 5alpha-cholestane-3beta,5,6beta-triol and 5,6alpha-epoxy-5alpha-cholestan-3alpha-ol in LDL, and the concentration of endothelin-1 (H: 0.9 +/- 0.2, N: 0.7 +/- 0.1 ng/ml) in plasma were increased in hypertensive patients. No differences were found for.NO derivatives between groups. These data suggest that an increase in cholesterol oxidation is associated with endothelium dysfunction in essential hypertension and oxidative stress, although.NO metabolite levels in plasma are not modified in the presence of elevated cholesterol oxides.
Assuntos
Endotélio Vascular/fisiopatologia , Hipertensão/fisiopatologia , Peroxidação de Lipídeos/fisiologia , Óxido Nítrico/sangue , Estresse Oxidativo/fisiologia , Vasodilatação/fisiologia , Adulto , Idoso , Disponibilidade Biológica , Estudos de Casos e Controles , LDL-Colesterol/química , LDL-Colesterol/metabolismo , Cromatografia , Endotelina-1/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Hipertensão/sangue , Masculino , Pessoa de Meia-IdadeRESUMO
Lipid and acute-phase protein alterations have been described in various infection diseases, and they have been recorded during the early stages of HIV infection. Lipid and acute-phase protein profiles also have been correlated with cellular immunological abnormalities. To document these correlations during HIV infection, we studied 75 HIV-infected patients and 26 HIV-negative controls. Patients were classified according to the criteria proposed by the Walter Reed Army Institute: as WR-1 (CD4 lymphocytes, 1154 +/- 268/mm3), WR-2 (CD4, 793 +/- 348/mm3) and WR3/4 (CD4, 287+/-75 mm3). Triglycerides, total cholesterol and HDL-cholesterol concentrations were measured by enzymatic methods. Immunoglobulins (IgA and IgG) and acute-phase proteins (haptoglobin, alpha1-acid glycoprotein, C-reactive protein and transferrin) were determined by immunonephelometry. Haptoglobin levels were significantly increased in HIV-positive patients and correlated with the progression of HIV-infection (controlAssuntos
Proteínas de Fase Aguda/metabolismo
, Infecções por HIV/sangue
, HIV-1
, Lipoproteínas/sangue
, Adulto
, Proteína C-Reativa/metabolismo
, Contagem de Linfócito CD4
, Estudos de Casos e Controles
, Colesterol/sangue
, HDL-Colesterol/sangue
, Feminino
, Haptoglobinas/metabolismo
, Humanos
, Imunoglobulina A/sangue
, Imunoglobulina G/sangue
, Masculino
, Orosomucoide/metabolismo
, Transferrina/metabolismo
, Triglicerídeos/sangue
RESUMO
Information is scarce on the prevalence of hepatitis B (HBV) and hepatitis C (HCV) among voluntary blood donors and patients infected with the human immunodeficiency virus (HIV) in Florianópolis, Brazil. A total of 2,678 serum samples from 2,583 blood donors and 95 HIV-infected patients, collected between April, 1994, and March, 1995, were examined for markers of HBV and HCV. All the samples were analyzed to detect HBV and HCV markers (HBsAg, anti-HBc, and anti-HCV). Hepatitis B and C prevalence among the studied blood donors reached 9.3% and 1.0%, respectively; 0.7% being seropositive for HBsAg and 9.2% for anti-HBc. It was also verified that 0.1% of blood donors were seropositive for HBsAg alone, 8.6% seropositive for the anti-HBc alone, and 0.6% presented a positive reaction for both of the HBV markers studied. Among HIV-infected patients, prevalence of 69.5% and 54.7% for hepatitis B and hepatitis C, respectively, were observed. Of these patients, 18.9% were seropositive for HBsAg, and 66.3% for the anti-HBc. The prevalence of a reaction for HBsAg alone, and for anti-HBc alone was 3.1% and 50.5%, respectively, for HIV-infected patients, whereas 15.8% were seropositive for both of the studied markers. HBV and HCV coinfection was 0.1% in blood donors, and 40% of those patients tested seropositive for HIV. Results show prevalence of HBV and HCV infection to be significantly greater among HIV-infected patients than among blood donors. These observations confirm the high frequency of HIV-infected patients exposure to these other viruses.