Lysophosphatidic acid (LPA) is a potent bioactive lipid component of oxidized low density lipoproteins (oxLDL). High concentrations of LPA have been detected in human atherosclerotic plaques. Our data has shown that LPA highly induces smooth muscle cell (SMC) migration. Cyr61, a matricellular protein, which also accumulates in human atherosclerotic plaques, has been implicated in the injury-induced neointimal formation. Smooth muscle cell migration is a key event in the development of atherosclerosis, and it contributes to the progressive growth of atherosclerotic lesions. Data generated by this study demonstrate that LPA markedly induces Cyr61 expression in mouse aortic smooth muscle cells (MASMC). We hypothesized that LPA-induced matricellular Cyr61 mediates LPA-induced MASMC migration. To date, little is known about the relationship between LPA and Cyr61 in smooth muscle cells; the signaling pathway leading to LPA-induced Cyr61 is unknown. Furthermore, whether Cyr61 contributes to LPA-induced cell migration is unrevealed. Our study demonstrates that LPA, by binding to LPA1 receptor, activates the intracellular signaling pathway leading to the activation of PKCdelta which in turn contributes to the increased expression of Cyr61 in MASMCs. Interestingly, we found that after LPA-induced Cyr61 mRNA has been translated into its protein intracellularly, the de novo synthesized proteins promptly accumulate in the Golgi apparatus and then translocalize to the extracellular matrix. Importantly, our data reveal a novel LPA/Cyr61 pathway in controlling MASMC migration. Understanding the mechanism underlying LPA induction of Cyr61 provides new insight into pathogenesis of atherosclerosis.

Traumatic brain injury (TBI) has become an increasingly unmet clinical need due to intense military conflicts worldwide. Directly impacted brain cells suffer massive death, with neighboring cells succumbing to progressive neurodegeneration accompanied by inflammatory and other secondary cell death events. Subsequent neurodegenerative events may extend to normal areas beyond the core of injury, thereby exacerbating the central nervous system’s inflammatory response to TBI. Recently CD-36 (cluster of differentiation 36/fatty acid translocase (FAT), a class B scavenger receptor of modified low-density lipoproteins (mLDLs) in macrophages, has been implicated in lipid metabolism, atherosclerosis, oxidative stress, and tissue injury in cerebral ischemia, and in certain neurodegenerative diseases. Accordingly, we proposed that CD-36 has a pivotal role in the neuroinflammatory cascade that further contributes to the pathology of TBI. First, we explored the neuroinflammatory role of CD-36 after acute and chronic stages of TBI. Second, we employed a neuroinflammatory model to test the therapeutic effect of the soluble receptor of advanced end-glycation product (sRAGE); previously shown to abrogate increased CD-36 expression in stroke. Third, we further examined ameliorating TBI related inflammation as a therapeutic pathway by combination of stem cell therapy and sRAGE. At acute stages of TBI, we observed brain co-localization of CD-36, monocyte chemoattractant protein 1 (MCP-1) and ionized calcium-binding adapter molecule 1 (Iba-1) on impacted cortical areas, significant increases of CD-36 and MCP-1 positive cells in the ipsilateral vs. contralateral hemispheres of TBI animals in acute, but no significant increases of Iba-1 expressing cells over time. In early acute stages of TBI immunoblotting showed overexpression of CD-36 in brain cortex when comparing ipsilateral and contralateral hemispheres vs. sham. Spleen CD-36 protein expression at acute post-TBI stages showed no significant difference between TBI and sham groups. In addition, immunohistochemistry revealed minimal CD-36 detection on the cortical area of impact on our chronic group. Spleen immunohistochemistry also showed co-localization of CD-36 and MCP-1 in the red pulp of spleen in acute stages of TBI animals when compared to sham. Ongoing ischemic and hyperlipidemic rodent models suggest that infiltrating monocytes/macrophages from the periphery are the major source of CD-36 in the post-ischemic brain. Likewise, CD-36 expressing monocytes in the spleen after TBI may suggest its role in peripheral immune response, which may exacerbates the inflammatory response after TBI. Therefore, CD-36 may play a key role as a pathological link between inflammation and TBI. Our results suggest an intimate involvement of CD-36 mediated inflammation in TBI, providing novel insights into the understanding of disease neuroinflammation and as a potent therapeutic target for TBI treatment. The critical timing (i.e., 24-48 hours) of CD-36 expression (from downregulation to upregulation) may signal the transition of functional effects of this immune response from pro-survival to cell death. This observed dynamic CD-36 expression also suggests the therapeutic window for TBI. The detection of CD-36 expression in brain areas proximal, as well as distal, to the site of impacted injury suggests its role in both acute and progressive evolution of TBI. CD-36 neuroinflammatory role has clinical relevance for treating patients who have suffered any TBI condition at acute and chronic stages.

The effects of oxidatively modified low density lipoprotein (oxLDL) on atherogenesis may be partly mediated by alterations in the production of nitric oxide (NO) by vascular cells. Lipid hydroperoxides (LOOH) and lysophosphatidylcholine (lysoPC) are the major primary products of LDL oxidation. The purpose of this study was to characterize the effects of oxLDL, LOOH and lysoPC on NO production and the expression of inducible nitric oxide synthase (iNOS) gene in lipopolysaccharide (LPS) stimulated macrophages. LDL was oxidized using an azo-initiator 2,2'-azobis (2- amidinopropane) HCl (ABAP) and octadecadienoic acid was oxidized by lipoxygenase to generate 13-hydroperoxyl octadecadienoic acid (13-HPODE). Our study showed that oxLDL markedly decreased the production of NO, the levels of iNOS protein and iNOS mRNA in LPS stimulated macrophages. The inhibition potential of oxLDL on NO production and iNOS gene expression depended on the levels of LOOH formed in oxLDL and was not due to oxLDL cytotoxicity. Furthermore, 13-HPODE markedly reduced NO production and iNOS protein levels, whereas lysoPC showed only slight reduction. The effects of 13-HPODE and lysoPC did not require an acetylated LDL carrier. Our results suggest that 13-HPODE is a much more potent inhibitor of NO production and iNOS gene expression than lysoPC in LPS stimulated RAW264.7 macrophages.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23802号 / 医博第4848号 / 新制||医||1058(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 永井 洋士, 教授 羽賀 博典, 教授 木村 剛 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Orientador: Wilson Nadruz Junior / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T13:58:57Z (GMT). No. of bitstreams: 1 Paim_LaydeRosane_M.pdf: 776318 bytes, checksum: f8e1046411d4b45041c7d37ca207a89e (MD5) Previous issue date: 2014 / Resumo: Estudos prévios mostram que indivíduos com lesão crônica na medula espinhal (LM) apresentam maior risco cardiovascular em comparação com indivíduos fisicamente normais. O presente estudo investigou a relação entre os níveis plasmáticos da lipoproteína de baixa densidade oxidada (LDLox), as metaloproteinases de matriz (MMP) e seus inibidores teciduais (TIMPs) e o remodelamento vascular em pacientes com LM, e o papel da atividade física nesta relação. Foram estudados 42 homens com LM (? 2 anos), [18 sedentários (S-LM) e 24 fisicamente ativos (A-LM)] e 16 homens fisicamente saudáveis por meio de análise clínica, antropométrica, laboratorial e de espessura íntima-média da carótida (EIM). Todos os participantes estudados eram normotensos, não diabéticos, não fumantes e normolipêmicos. As concentrações plasmáticas de LDLox, MMP-2, MMP-8, MMP-9, TIMP-1 e TIMP-2 foram determinados por ensaio imunoenzimático (ELISA). Os resultados mostraram que a EIM da carótida, razão EIM/diâmetro e as concentrações de LDLox dos A-LM e dos indivíduos fisicamente normais não foram diferentes estatisticamente. Por outro lado, indivíduos com S-LM apresentaram maior EIM, razão EIM/diâmetro e concentrações aumentados de LDLox em comparação com A-LM (p<0,01, p<0,001 e p=0,01, respectivamente) e indivíduos controles (p<0,001 para todos). Os resultados da análise de correlação bivariada, incluindo todos os indivíduos com LM, demonstrou que o EIM de carótida e a razão EIM/diâmetro se correlacionaram apenas com LDLox, MMP-8 e com a relação MMP-8/TIMP-1. Além disso, a análise de regressão ajustada para a presença ou não de atividade física e idade mostrou que a LDLox foi associada à EIM carotídea e com a relação EIM/diâmetro, enquanto que MMP-8 foi associado com o índice EIM/diâmetro em indivíduos com LM. Em conclusão, as concentrações plasmáticas de LDLox e MMP-8 estão associados com aterosclerose carotídea e há interação entre a inatividade física, aterosclerose e LDLox em indivíduos com LM / Abstract: Previous reports have indicated that subjects with chronic spinal cord injury (SCI) exhibit increased cardiovascular risk compared to able-bodied individuals. This study investigated the relationship between plasmatic oxidized low-density lipoprotein (OxLDL), matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) levels and vascular remodeling in SCI subjects and the role of physical activity in this regard. We studied 42 men with chronic (?2 years) SCI [18 sedentary (S-SCI) and 24 physically active (PA-SCI)] and 16 able-bodied men by clinical, anthropometric, laboratory, and carotid intima-media thickness (IMT) analysis. All enrolled subjects were normotensive, non-diabetics, non-smokers and normolipemic. Plasmatic OxLDL, MMP-2, MMP-8, MMP-9, TIMP-1 and TIMP-2 levels were determined by enzyme-linked immunosorbent assay. The results showed that carotid IMT, IMT/diameter ratio and OxLDL levels of PA-SCI and able-bodied subjects were statistically similar. Conversely, S-SCI subjects exhibited higher IMT, IMT/diameter ratio and OxLDL levels compared to PA-SCI (p<0.01, p<0.001 and p=0.01, respectively) and able-bodied (p<0.001 for all) individuals. Results of bivariate correlation analysis including all injured subjects showed that carotid IMT and IMT/diameter ratio only correlated with OxLDL, MMP-8 and MMP-8/TIMP-1 ratio. Further stepwise regression analysis adjusted for the presence or not of physical activity and age showed that OxLDL was associated with carotid IMT and IMT/diameter ratio, while MMP-8 was associated with IMT/diameter ratio in SCI individuals. In conclusion, plasmatic OxLDL and MMP-8 levels are associated with carotid atherosclerosis and there is an interaction among physical inactivity, atherosclerosis and OxLDL in SCI individuals / Mestrado / Clinica Medica / Mestra em Clínica Médica

This study investigated whether oxLDL and/or angiotensin (Ang) II signaling pathways mediate traffic-generated air pollution- exposure induced alterations in blood-brain barrier (BBB) integrity and permeability in a healthy wild type (C57Bl/6) mouse model; additionally, whether these outcomes are exacerbated by a high fat-diet investigated. An environmentally relevant concentration of a mixture of vehicle engine exhaust (MVE) was used. To investigate the hypotheses, 12 wk old male C57Bl/6 mice on either a high fat (HF) or low fat (LF) diet were randomly assigned to inhalational exposure of either filtered-air (FA) or 30 µg PM/m3 diesel exhaust + 70 µg PM/m3 gasoline exhaust (MVE) for 6 hr/day for 30 days. Additionally, we examined mechanisms involved in MVE-mediated alterations BBB integrity using a novel BBB co-culture in vitro model, consisting of mouse primary cerebral vascular endothelial cells on an apical transwell and astrocytes in the basal compartment, which was treated with plasma from the mice on our exposure study. Our in vivo exposure study results showed that MVE inhalation resulted in increased circulating plasma oxLDL and Ang II, compared to FA controls. Additionally, we observed increased cerebral microvascular expression of oxLDL receptors, LOX-1 and CD-36, and Ang II receptor subtype 1 (AT1) in MVE-exposed C57Bl/6 mice, which was further exacerbated with consumption of an HF diet. Increased signaling of both Ang II and oxLDL was associated with decreased BBB integrity, as evidenced by the concurrent reduction in expression of tight junction (TJ) protein claudin-5 and increased permeability of sodium fluorescein (Na-F) from the blood into the cerebral parenchyma. Our results suggest that possible mechanisms involved in oxLDL and/or Ang II-mediated alterations in BBB integrity include oxidative stress and upregulated expression and activity of matrix metalloproteinase (MMP)-9, which is associated with degradation of TJ proteins in the BBB. Our in vitro BBB co-culture results confirm our in vivo findings, as we observe increased BBB permeability (TEER) and decreased integrity (decreased expression of TJ proteins) in the endothelial (apical) layer when treated with plasma from MVE-exposed mice, which was further exacerbated when treated with plasma from MVE-exposed mice on an HF diet. Pre-treatment of the endothelial cells with the AT1 receptor antagonist, Losartan, prior to applying plasma, resulted in attenuation of the alterations observed in endothelial integrity in the BBB co-culture treated with plasma from either MVE+LF or MVE+HF animals. These results suggest Ang II – AT1 signaling mediate, at least in part, the alterations in the BBB integrity observed after exposure to MVE. Moreover, we observed that treatment of the endothelial (apical) layer with plasma from MVE-exposed animals resulted in increased production of inflammatory mediators interleukin-6 (IL-6) and transforming growth factor-β in the astrocyte media (basal compartment). Additionally, these same astrocytes also displayed increased production of angiotensin-converting enzyme (ACE) and also AT1 receptor mRNA expression, while showing decreased expression of the aryl hydrocarbon receptor (AhR) and glutathione peroxidase (GPx). Collectively, these results suggest that exposure to the ubiquitous environmental air pollutant, vehicle engine emissions, results in increased oxLDL and Ang II signaling in the cerebral microvasculature, which is associated with decreased vessel integrity and increased oxidative stress and inflammatory signaling in the CNS. The observed detrimental outcomes are even further exacerbated when coupled with the consumption of an HF diet.

C-reactive protein (CRP) is secreted by hepatocytes as a pentameric molecule made up of identical monomers, circulates in the plasma as pentamers, and localizes in atherosclerotic lesions. In some cases, localized CRP was detected by using monoclonal antibodies that did not react with native pentameric CRP but were specific for isolated monomeric CRP. It has been reported that, once CRP is bound to certain ligands, the pentameric structure of CRP is altered so that it can dissociate into monomers. Accordingly, the monomeric CRP found in atherosclerotic lesions may be a stationary, ligand-bound, by-product of a ligand-binding function of CRP. CRP binds to modified forms of low-density lipoprotein (LDL). The binding of CRP to oxidized LDL requires acidic pH conditions; the binding at physiological pH is controversial. The binding of CRP to enzymatically-modified LDL occurs at physiological pH; however, the binding is enhanced at acidic pH. Using enzymatically-modified LDL, CRP has been shown to prevent the formation of enzymatically-modified LDL-loaded macrophage foam cells. CRP is neither pro-atherogenic nor atheroprotective in ApoE-/-and ApoB100/100Ldlr-/-murine models of atherosclerosis, except in one study where CRP was found to be slightly atheroprotective in ApoB100/100Ldlr-/-mice. The reasons for the ineffectiveness of human CRP in murine models of atherosclerosis are not defined. It is possible that an inflammatory environment, such as those characterized by acidic pH, is needed for efficient interaction between CRP and atherogenic LDL during the development of atherosclerosis and to observe the possible atheroprotective function of CRP in animal models.

<p>In patients with unstable coronary artery disease (CAD) biochemical markers are emerging as useful tools in clinical management. In this thesis we studied the use of markers of low-density lipoprotein (LDL) oxidation and renal function.</p><p>Our study populations consisted of unstable CAD patients included in the Fast Revascularisation during Instability in Coronary artery disease (FRISC)-II trial and healthy controls. Patients were followed for 2 years regarding death and myocardial infarction (MI).</p><p>Using receiver operating characteristic curve analysis, we found that oxidized low-density lipoprotein (OxLDL), especially when combined with high-density lipoprotein, compared to traditionally measured lipids/lipoproteins, and a new lipoprotein marker, lipoprotein associated-phospholipase A2, was better at discriminating between healthy controls and CAD patients. In patients, OxLDL was found to be an independent prognostic marker associated with an increased risk of MI, of particular use in patients with no evidence of myocardial necrosis. </p><p>In our study on the effects of an early invasive treatment strategy in unstable CAD patients with mild to moderate renal dysfunction (i.e. creatinine clearance <90mL/min) we found that in patients randomized to invasive treatment, the rates of death/MI and MI alone were significantly lower than in patients randomized to non-invasive treatment. In patients treated invasively, no detrimental effects were seen on renal function at follow-up at 6 months. </p><p>In healthy controls, we investigated new markers of renal (cystatin C) and cardio-renal function (N-terminal probrain natriuretic peptide, [NT-proBNP]) regarding reference levels and physiological determinants. We found that cystatin C is influenced by age whereas NT-proBNP is influenced by age and gender.</p><p>Our studies suggest that OxLDL and renal dysfunction are associated with a poor prognosis in unstable CAD patients and that these markers demonstrate potential for clinical use. In the search for new markers related to renal function we have contributed with reference levels of cystatin C and NT-proBNP. </p>

In patients with unstable coronary artery disease (CAD) biochemical markers are emerging as useful tools in clinical management. In this thesis we studied the use of markers of low-density lipoprotein (LDL) oxidation and renal function. Our study populations consisted of unstable CAD patients included in the Fast Revascularisation during Instability in Coronary artery disease (FRISC)-II trial and healthy controls. Patients were followed for 2 years regarding death and myocardial infarction (MI). Using receiver operating characteristic curve analysis, we found that oxidized low-density lipoprotein (OxLDL), especially when combined with high-density lipoprotein, compared to traditionally measured lipids/lipoproteins, and a new lipoprotein marker, lipoprotein associated-phospholipase A2, was better at discriminating between healthy controls and CAD patients. In patients, OxLDL was found to be an independent prognostic marker associated with an increased risk of MI, of particular use in patients with no evidence of myocardial necrosis. In our study on the effects of an early invasive treatment strategy in unstable CAD patients with mild to moderate renal dysfunction (i.e. creatinine clearance &lt;90mL/min) we found that in patients randomized to invasive treatment, the rates of death/MI and MI alone were significantly lower than in patients randomized to non-invasive treatment. In patients treated invasively, no detrimental effects were seen on renal function at follow-up at 6 months. In healthy controls, we investigated new markers of renal (cystatin C) and cardio-renal function (N-terminal probrain natriuretic peptide, [NT-proBNP]) regarding reference levels and physiological determinants. We found that cystatin C is influenced by age whereas NT-proBNP is influenced by age and gender. Our studies suggest that OxLDL and renal dysfunction are associated with a poor prognosis in unstable CAD patients and that these markers demonstrate potential for clinical use. In the search for new markers related to renal function we have contributed with reference levels of cystatin C and NT-proBNP.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Atherosclerosis is a chronic inflammatory disease that is characterized by the accumulation of lipids and fibrous elements in the intima layer of arteries of medium and large caliber. The cardiovascular diseases resulting from atherosclerosis, among them acute myocardial infarction and stroke are the leading cause of mortality and morbidity worldwide. Oxidative stress and oxidative modification of low density lipoprotein (LDL) have an important role in the development of this disease and so the inclusion of antioxidants in the diet may prevent the progression of atherosclerosis. The carotenoid norbixin (NBX), which is found in annatto seeds, have excellent antioxidant activity as demonstrated in several models of oxidative damage. In this context, the objective of this study was to evaluate the antioxidant, anti-inflammatory and antiatherogenic potential of NBX in a model of atherosclerosis in rabbits. Male New Zealand rabbits received regular chow (control) or an atherogenic diet (0.5% cholesterol) alone or supplemented with NBX (10, 30 or 100 mg/kg) for 60 days. The antioxidant enzyme activities, lipid profiles, oxidative stress and inflammatory markers and histopathological status were evaluated in the serum or aortic tissue. The atherogenic diet increased serum lipids, oxidized low-density lipoprotein (LDLox) levels and oxidized low-density lipoprotein antibody (LDLoxAB) levels, in addition to inducing lipid and protein oxidation in the aortic tissue. Supplementation with NBX caused 35% reduction in the levels of LDLoxAB, 69% in the levels of LDLox, 27% in the levels of TBARS and 46% in the levels of protein carbonyl induced by the atherogenic diet, besides increasing up to 88% the HDL levels. In atherosclerotic rabbits, the non-protein thiol group content and enzymatic activity of the antioxidants superoxide dismutase, catalase, glutathione reductase and thioredoxin reductase were increased in aortic tissue, whereas paraoxonase activity was reduced in serum. Supplementation with NBX restored up to 41% the increased levels of NPSH, 37% SOD activity, 45% CAT activity, 66% GR activity, 50% TrxR-1 activity induced by the atherogenic diet. NBX also restored 15% of PON1 activity inhibited by the atherogenic diet. The atherogenic diet also increased the serum levels of inflammatory markers and the ratio of the intima area to the media area in the aortic arch; these changes were not prevented by NBX. Thus, NBX supplementation improved the lipid profile, decreased oxidative stress and prevented changes in paraoxonase activity and in the antioxidant system in hypercholesterolemic rabbits, but did not prevent the formation of atherosclerotic plaques. These results support a beneficial role of NBX in the treatment of atherosclerosis by preventing oxidative events and by restoring antioxidant enzyme activity and paraoxonase activity. / A aterosclerose é uma doença inflamatória crônica caracterizada pelo acúmulo de lipídeos e elementos fibrosos na túnica intima das artérias de médio e grande calibre. As doenças cardiovasculares decorrentes da aterosclerose, dentre elas o infarto agudo do miocárdio e o acidente vascular cerebral são a principal causa de mortalidade e morbidade mundial. O estresse oxidativo e a modificação oxidativa da lipoproteína de baixa densidade (LDL) possuem um papel importante no desenvolvimento dessa doença. Por este motivo a inclusão de antioxidantes na dieta poderia impedir a progressão da aterosclerose. O carotenóide norbixina (NBX), presente nas sementes de urucum, possui excelente atividade antioxidante já demonstrada em diversos modelos de dano oxidativo. Nesse contexto, o objetivo deste trabalho foi avaliar o potencial antioxidante, anti-inflamatório e antiaterogênico da NBX em um modelo de aterosclerose em coelhos. Coelhos Nova Zelândia machos receberam ração regular (controle) ou uma dieta aterogênica (0,5% de colesterol) sozinha ou suplementada com NBX (10, 30 ou 100 mg/kg) por 60 dias. A atividade das enzimas antioxidantes, o perfil lipídico, os marcadores de estresse oxidativo e inflamação e as alterações histopatológicas foram avaliados no soro ou tecido aórtico dos coelhos hipercolesterolêmicos. A dieta aterogênica aumentou os níveis séricos de lipídios, de lipoproteína de baixa densidade oxidada (LDLox) e de anticorpos contra lipoproteína de baixa densidade oxidada (LDLoxAB), além de induzir a oxidação de lipídios e proteína no tecido aórtico. A suplementação com NBX reduziu em até 35% o aumento dos níveis de LDLoxAB, 69% dos níveis de LDLox, 27% dos níveis de TBARS e 46% dos níveis de proteínas carboniladas induzidos pela dieta aterogênica, além de aumentar em até 88% os níveis de HDL. Nos coelhos ateroscleróticos ocorreu uma elevação no conteúdo de grupos tiólicos não-protéicos (NPSH) e na atividade das enzimas antioxidantes superóxido dismutase (SOD), catalase (CAT), glutationa redutase (GR) e tioredoxina redutase (TrxR-1) no tecido aórtico, enquanto a atividade da enzima paraoxonase (PON1) foi reduzida no soro. A suplementação com NBX reduziu em até 41% o aumento dos níveis de NPSH, 37% da atividade da SOD, 45% da atividade da CAT, 66% da atividade da GR e 50% da atividade da TrxR-1 induzidos pela dieta aterogênica. A NBX também restaurou em 15% a atividade da PON1 inibida pela dieta aterogênica. A dieta aterogênica também aumentou os níveis séricos de marcadores inflamatórios e a relação entre a área da íntima e da média no arco aórtico e essas mudanças não foram prevenidas pela NBX. Assim, a suplementação com NBX melhorou o perfil lipídico, diminuiu o estresse oxidativo e impediu mudanças na atividade da paraoxonase e no sistema antioxidante em coelhos hipercolesterolêmicos, mas não impediu a formação de placas ateroscleróticas. Esses resultados indicam um papel benéfico da NBX no tratamento de aterosclerose, impedindo eventos oxidativo e restaurando a atividade das enzimas antioxidantes e da enzima paraoxonase.