Tableau d’honneur de la Faculté des études supérieures et postdoctorales, 2013-2014. / Un stress oxydatif survient lorsqu’il y a surproduction de dérivés actifs de l’oxygène par rapport aux défenses antioxydantes. Ce déséquilibre est associé, entre autres, à la prééclampsie, une pathologie de la grossesse. Les F2-isoprostanes regroupent soixante-quatre isomères issus de la peroxydation de l’acide arachidonique. Ceux-ci sont reconnus comme étant les biomarqueurs les plus fiables du stress oxydatif in vivo. Une méthode d’analyse par chromatographie liquide couplée à la spectrométrie de masse en tandem pour le dosage de sept isomères de F2-isoprostanes dans des échantillons de plasma, de sang et de membranes érythrocytaires a été mise au point et validée. Les F2-isoprostanes dans le plasma ont été corrélés positivement avec plusieurs acides gras trans plasmatiques au troisième trimestre de la grossesse. Contre toute attente, les F2-isoprostanes du plasma, du sang et des membranes érythrocytaires sont moins abondants en prééclampsie par rapport aux contrôles en fin de grossesse. / An oxidative stress is defined as an imbalance between the production of reactive oxygen species and antioxidant defenses of the organism. This imbalance has been associated with preeclampsia, a pathology of the mid-to-late pregnancy. Peroxidation of arachidonic acid generates sixty-four isomers of F2-isoprostanes. The latter are recognized as the most reliable biomarkers of oxidative stress in vivo. A method using liquid chromatography tandem mass spectrometry (HPLC-MS/MS) for the determination of seven isomers of F2-isoprostanes in plasma samples, whole blood and erythrocyte membranes has been developed and validated. The F2-isoprostanes correlated positively with several trans fatty acids in plasma at end of the pregnancy. Unexpectedly, F2-isoprostanes were less abundant in preeclampsia than in control pregnancies at the third trimester.

The aims were to investigate oxidative stress (I), angiogenesis (II) and inflammation (III-IV) in healthy women during pregnancy and postpartum. Oxidative stress was estimated by measurement of 8-iso-PGF2α and the antioxidants α- and γ-tocopherol. The angiogenic factors PlGF, VEGF-A and the antiangiogenic factor sFlt1 were measured to estimate angiogenesis. PTX3, IL-6, TNF-α and a PGF2α metabolite were measured to estimate inflammation. Out of 52 included women, 15 had minor pregnancy complications and 37 were classified as normal. In study III data from all 52 women were used. For the other studies (I, II and IV) only data from the 37 women with normal pregnancy were used. Pregnancy was associated with increased levels of 8-iso-PGF2α with advancing gestational age. The median postpartum value corresponded to values observed in early gestation and a significant decrease was observed from late pregnancy to postpartum. Lipid-adjusted α- and γ-tocopherol levels decreased with advancing gestational age (I). PlGF increased from early pregnancy until weeks 29–30 and thereafter decreased until week 40. sFlt1 levels were relatively constant until weeks 29–30, when they increased, reaching a peak at weeks 39–40. Postpartum levels were low. The sFlt1:PlGF ratio decreased from weeks 9–12, was constantly low from weeks 19–20 to 37–38 and then increased to weeks 39–40. VEGF-A was detectable in only 8 % of the samples during pregnancy and in 64 % postpartum (II). There was a continuous increase of PTX3 as pregnancy progressed. The increase was most evident after week 31 with the highest levels just before delivery (III). IL-6 increased throughout pregnancy and remained high postpartum. No change in TNF-α could be seen with advancing gestational age or postpartum. The PGF2α metabolite levels increased throughout pregnancy and decreased postpartum (IV). In conclusion, normal pregnancy is associated with mild oxidative stress and inflammation. This might have physiological effects for normal pregnancy development. By delineating how these mediators of oxidative stress, angiogenesis and inflammation fluctuate throughout normal pregnancy and postpartum, we have established a reference for studies of these factors in pregnancy complications.

Orientador: Patrícia Moriel / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T00:37:20Z (GMT). No. of bitstreams: 1 Freitas_BetaniadeJesuseSilvadeAlmendra_D.pdf: 2657931 bytes, checksum: d4646bbc60ccc13e11ca7d806b4f75dc (MD5) Previous issue date: 2014 / Resumo: Estudos acerca da influência do estresse oxidativo sobre o equilíbrio cutâneo, sobretudo por seus efeitos devastadores sobre a integridade da pele, são essenciais para a proposição de estratégias de intervenção preventivas para o desenvolvimento do câncer de pele. O objetivo do estudo foi comparar o estresse oxidativo de indivíduos que tiveram e não tiveram câncer de pele não melanoma e avaliar o efeito da suplementação combinada de vitaminas C, E e mineral Zinco no estresse oxidativo de indivíduos que apresentaram a doença. O estudo foi dividido em duas fases: a fase 1 foi um estudo transversal com controles, cuja população foi constituída por pessoas saudáveis (n = 24) e o grupo caso constituído por indivíduos que apresentaram câncer de pele não melanoma já submetidas a tratamento cirúrgico (n = 60). E a fase 2, um ensaio clínico randomizado e duplo cego, no qual os pacientes do grupo caso foram randomizados em dois subgrupos: grupo placebo (n = 34) e grupo suplementado (n = 26) com 50 mg de vitamina C, 60 mg de vitamina E e 40 mg de Zinco durante 8 semanas. As amostras de sangue dos sujeitos foram obtidas no período basal e após intervenção para a avaliação dos biomarcadores de estresse oxidativo (F2-isoprostano, nitrito, substâncias reativas ao ácido tiobarbitúrico (TBARS) e capacidade antioxidante total). O consumo alimentar habitual e o estado nutricional dos sujeitos foram avaliados. Para identificação dos fatores associados ao câncer de pele foi utilizada a análise de regressão logística univariada e multivariada. O nível de significância adotado para este estudo foi de 5%. A maioria dos pacientes estudados foram do sexo feminino com idade superior a 50 anos. Os pacientes do grupo caso apresentaram mais elevadas concentrações séricas dos biomarcadores de estresse oxidativo, sendo que as concentrações de F2-isoprostano estavam significativamente mais elevadas em comparação com os controles. Após suplementação não houve diferença estatística entre os grupos placebo e suplementado em relação aos marcadores de estresse oxidativo. A idade e o F2-isoprostano podem ser marcadores de risco para o câncer de pele não melanoma, a cada ano a mais para o fator idade aumenta em 12% a chance de câncer e cada unidade a mais na medida do marcador aumenta em 4% a chance de câncer. Os resultados mostraram prevalência de sobrepeso no grupo controle com diferença estatística significativa em relação ao grupo caso. As concentrações dietéticas dos minerais antioxidantes zinco, cobre e selênio do grupo caso foram estatisticamente inferiores em relação aos controles e não houve diferença estatística nas concentrações dietéticas dos nutrientes antioxidantes entre os grupos suplementado e placebo. Este estudo sugere que pessoas diagnosticadas com câncer de pele não melanoma e que no momento da realização da pesquisa não mais apresentavam a doença, mostravam elevado estresse oxidativo, quando comparadas a pessoas saudáveis. A suplementação de antioxidantes pelo período de tempo realizado no trabalho não provocou redução significativa nas concentrações dos marcadores de estresse oxidativo dos pacientes. O estudo ainda sugere que o marcador de estresse oxidativo F2-isoprostano pode ser utilizado como um fator de risco para o desenvolvimento do câncer de pele não melanoma / Abstract: Studies investigating the influence of oxidative stress on skin homeostasis, especially for its devastating effects on skin integrity, are essential for the development of preventive intervention strategies for skin cancer. The goal of this study was to compare the concentrations of oxidative stress biomarkers in blood between individuals with and without non-melanoma skin cancer and evaluate the effect of combined supplementation with vitamins C, E, and the mineral zinc on oxidative stress in skin-cancer patients. The study was divided into two stages: stage 1 was cross-sectional study with controls, whose population consisted of healthy individuals (n = 24) and the case group included individuals who had non-melanoma skin cancer undergoing surgery (n = 60). And the second phase a randomized, double blind clinical trial where patients in the case group were randomized into two subgroups: placebo (n =34) and a supplemented group (n = 26) who received 50 mg of vitamin C, 60 mg of vitamin E, and 40 mg of zinc for 8 wk. Blood samples were taken from the subjects before and after intervention to evaluate levels of oxidative stress biomarkers (F2-isoprostane, nitrite, thiobarbituric acid reactive substances (TBARS) and total antioxidant capacity. The usual food consumption and nutritional state of the subjects were also evaluated. Multivariate and univariate logistics regression analysis were used to identify factors associated with the development of skin cancer. The level of significance adopted for this study was 5%. The majority of participants were women over the age of 50. The patients in the case group had higher serum concentrations of oxidative stress biomarkers, and the levels of F2-isoprostane were significantly higher than the controls. After antioxidant supplementation there was no statistical difference in the markers of oxidative stress among the placebo and supplemented groups. Age and F2-isoprostane may be effective biomarkers for estimating the risk of non-melanoma skin cancer development. Moreover, the risk of cancer increases with age at a rate of 12% per year, while an increase in concentration of these biomarker in blood increases the risk of cancer by 4%. These results showed a prevalence of excess weight in the control group with significant statistical difference from the case group. The dietary intake of the mineral antioxidants zinc, copper, and selenium of the case group were significantly lower than the control group, and there was no statistical difference in the dietary intake of the antioxidant nutrients among the supplemented and placebo groups. This study suggests that people diagnosed with non-melanoma skin cancer and those in remission at the time of the study, exhibited higher concentration oxidative stress than healthy individuals. The antioxidant supplementation by period the work performed did not cause significant reduction in serum concentrations of oxidative stress biomarkers of the patients. The results suggest that the concentration of the oxidative stress biomarker, F2-isoprostane, may serve as risk factor for non-melanoma skin cancer development / Doutorado / Ciencias Biomedicas / Doutora em Ciências Médicas

<p>Lipid peroxidation is thought to be an important factor in the pathophysiology of a number of diseases and in the process of ageing, but its measurement <i>in vivo</i> has been difficult. The aim of this thesis was to evaluate methods for measurement of lipid peroxidation <i>in vivo</i> that are suitable for clinical investigations, and to apply these methods in animal and human studies investigating basal conditions and situations associated with increased lipid peroxidation.</p><p>The ferrous oxidation in xylenol orange assay for quantification of total plasma lipid hydroperoxides was re-evaluated regarding sample handling and storage. It was shown to be a useful tool for analyses of fresh but not stored plasma samples.</p><p>A methodology for measurement of the total amount (sum of free and esterified) of an F₂-isoprostane, 8-iso-prostaglandin F_2α, in tissues using alkaline hydrolysis in combination with an existing radioimmunoassay was developed. High levels of 8-iso-prostaglandin F_2α in rat liver tissue were quantified by this technique both at basal conditions and in an experimental model of increased lipid peroxidation induced by carbon tetrachloride.</p><p>Supplementation with vitamin E to rats decreased both non-enzymatic and enzymatic lipid peroxidation as measured by 8-iso-prostaglandin F_2α and a major prostaglandin F_2α metabolite. This was verified both in the urine at basal conditions, and in the urine and liver tissue after carbon tetrachloride induced lipid peroxidation.</p><p>In a randomised cross-over study in humans, a rapeseed oil-based diet with an increased proportion of easily oxidised polyunsaturated fatty acids was compared to a control diet rich in saturated fats. The rapeseed oil-based diet did not seem to increase the degree of lipid peroxidation in plasma and urine as measured by 8-iso-prostaglandin F_2α, hydroperoxides and malondialdehyde, presumably due to a sufficient content of antioxidants in the rapeseed oil diet.</p><p>In conclusion, the simultaneous measurement of several biomarkers of lipid peroxidation is a promising approach for future studies investigating the possible role of lipid peroxidation <i>in vivo</i> under basal conditions and in the pathology of diseases.</p>

<p>Maternal type 1 diabetes is associated with an increased risk for foetal malformations. The mechanism by which diabetes is teratogenic is not fully known. Previous studies have demonstrated that radical oxygen species can contribute to the teratogenicity of glucose and diabetes. The aim of the present work was to study different aspects of free radical damage and antioxidant defence in experimental diabetic pregnancy.</p><p> The activity of the antioxidant enzyme catalase and the mRNA levels of antioxidant enzymes in embryos of normal and diabetic rats of two strains were measured. The catalase activity was higher in embryos of a malformation-resistant strain than in a malformation-prone strain, the difference increased further when the mother was diabetic. Maternal diabetes increased embryonic mRNA levels of catalase and manganese superoxide dismutase in the malformation-resistant strain, but not in the malformation-prone strain. Embryos of the malformation-prone rat thus had lower antioxidative defence than embryos of the malformation-resistant strain.</p><p> Administration of either vitamin E or vitamin C has previously been shown to protect embryos from maldevelopment in experimental diabetic pregnancy. The vitamins were used together in this thesis to yield protection in both the lipid and aqueous phase. The protective effect was not higher than what had been achieved using the vitamins individually. No synergistic effect was thus found using the two antioxidants together. </p><p> The urinary excretion of the lipid peroxidation marker 8-iso-PGF_2á was increased in pregnant dia-betic rats compared with non-diabetic controls, as was the plasma content of carbonylated proteins. Carbonylated proteins and TBARS concentrations were increased in foetal livers in diabetic pregnancy. However, no increased concentration of 8-iso-PGF_2á was found in the amniotic fluid of pregnant diabetic rats. Both lipids and proteins were thus oxidatively modified in experimental diabetic pregnancy. It is concluded that experimental diabetic pregnancy is associated with increased oxidative stress and that the embryonic antioxidant defence is likely to be of importance for normal development in a diabetic environment.</p>

<p>Numerous diseases are associated with reduced antioxidant defence and oxidative stress. The antioxidant defence includes dietary and endogenous antioxidants and involves complex interactions between them. The effects of dietary factors on antioxidant status and oxidative stress of healthy humans were investigated in the studies described in this thesis. Assays of plasma antioxidant capacity encompass interactions between various antioxidants. Although uric acid has an unclear function as an antioxidant, it is a major determinant of antioxidant capacity. We measured antioxidant capacity in the presence and absence of uric acid to provide more information on the application of measures of antioxidant capacity. Individuals with high dietary intakes of various antioxidants and antioxidant rich foods, especially when combined, had higher plasma antioxidant capacities than those with lower antioxidant intakes. However, there were no associations between dietary intake of antioxidants or antioxidant rich foods and the plasma concentration of F₂-isoprostanes, which is considered a reliable biomarker for oxidative stress. Intakes of various doses of a mixture of bilberry juice and black tea, rich in flavonoids for four weeks, increased antioxidant capacity in some groups, but urine levels of F₂-isoprostanes were not affected. There were substantial individual variations in responses to the drinks related to baseline antioxidant capacity. Supplementation with eicosapentaenoic acid and docosahexaenoic acid decreased the plasma levels of F₂-isoprostanes, but not prostaglandin F_2α formation or antioxidant capacity. </p><p>It was concluded that a high intake of foods rich in antioxidants is related to improved antioxidant status. After intake of foods rich in antioxidants, the antioxidant status may increase, but with considerable individual variation in the responses, which warrants further investigation. Lipid peroxidation <i>in vivo</i> is not easily affected by dietary antioxidants in healthy humans. Although n-3 fatty acids are highly unsaturated, they reduce nonenzymatic free radical-catalyzed lipid peroxidation, but not enzymatic lipid peroxidation.</p>

Lipid peroxidation is thought to be an important factor in the pathophysiology of a number of diseases and in the process of ageing, but its measurement in vivo has been difficult. The aim of this thesis was to evaluate methods for measurement of lipid peroxidation in vivo that are suitable for clinical investigations, and to apply these methods in animal and human studies investigating basal conditions and situations associated with increased lipid peroxidation. The ferrous oxidation in xylenol orange assay for quantification of total plasma lipid hydroperoxides was re-evaluated regarding sample handling and storage. It was shown to be a useful tool for analyses of fresh but not stored plasma samples. A methodology for measurement of the total amount (sum of free and esterified) of an F2-isoprostane, 8-iso-prostaglandin F2α, in tissues using alkaline hydrolysis in combination with an existing radioimmunoassay was developed. High levels of 8-iso-prostaglandin F2α in rat liver tissue were quantified by this technique both at basal conditions and in an experimental model of increased lipid peroxidation induced by carbon tetrachloride. Supplementation with vitamin E to rats decreased both non-enzymatic and enzymatic lipid peroxidation as measured by 8-iso-prostaglandin F2α and a major prostaglandin F2α metabolite. This was verified both in the urine at basal conditions, and in the urine and liver tissue after carbon tetrachloride induced lipid peroxidation. In a randomised cross-over study in humans, a rapeseed oil-based diet with an increased proportion of easily oxidised polyunsaturated fatty acids was compared to a control diet rich in saturated fats. The rapeseed oil-based diet did not seem to increase the degree of lipid peroxidation in plasma and urine as measured by 8-iso-prostaglandin F2α, hydroperoxides and malondialdehyde, presumably due to a sufficient content of antioxidants in the rapeseed oil diet. In conclusion, the simultaneous measurement of several biomarkers of lipid peroxidation is a promising approach for future studies investigating the possible role of lipid peroxidation in vivo under basal conditions and in the pathology of diseases.

Maternal type 1 diabetes is associated with an increased risk for foetal malformations. The mechanism by which diabetes is teratogenic is not fully known. Previous studies have demonstrated that radical oxygen species can contribute to the teratogenicity of glucose and diabetes. The aim of the present work was to study different aspects of free radical damage and antioxidant defence in experimental diabetic pregnancy. The activity of the antioxidant enzyme catalase and the mRNA levels of antioxidant enzymes in embryos of normal and diabetic rats of two strains were measured. The catalase activity was higher in embryos of a malformation-resistant strain than in a malformation-prone strain, the difference increased further when the mother was diabetic. Maternal diabetes increased embryonic mRNA levels of catalase and manganese superoxide dismutase in the malformation-resistant strain, but not in the malformation-prone strain. Embryos of the malformation-prone rat thus had lower antioxidative defence than embryos of the malformation-resistant strain. Administration of either vitamin E or vitamin C has previously been shown to protect embryos from maldevelopment in experimental diabetic pregnancy. The vitamins were used together in this thesis to yield protection in both the lipid and aqueous phase. The protective effect was not higher than what had been achieved using the vitamins individually. No synergistic effect was thus found using the two antioxidants together. The urinary excretion of the lipid peroxidation marker 8-iso-PGF2á was increased in pregnant dia-betic rats compared with non-diabetic controls, as was the plasma content of carbonylated proteins. Carbonylated proteins and TBARS concentrations were increased in foetal livers in diabetic pregnancy. However, no increased concentration of 8-iso-PGF2á was found in the amniotic fluid of pregnant diabetic rats. Both lipids and proteins were thus oxidatively modified in experimental diabetic pregnancy. It is concluded that experimental diabetic pregnancy is associated with increased oxidative stress and that the embryonic antioxidant defence is likely to be of importance for normal development in a diabetic environment.

Numerous diseases are associated with reduced antioxidant defence and oxidative stress. The antioxidant defence includes dietary and endogenous antioxidants and involves complex interactions between them. The effects of dietary factors on antioxidant status and oxidative stress of healthy humans were investigated in the studies described in this thesis. Assays of plasma antioxidant capacity encompass interactions between various antioxidants. Although uric acid has an unclear function as an antioxidant, it is a major determinant of antioxidant capacity. We measured antioxidant capacity in the presence and absence of uric acid to provide more information on the application of measures of antioxidant capacity. Individuals with high dietary intakes of various antioxidants and antioxidant rich foods, especially when combined, had higher plasma antioxidant capacities than those with lower antioxidant intakes. However, there were no associations between dietary intake of antioxidants or antioxidant rich foods and the plasma concentration of F2-isoprostanes, which is considered a reliable biomarker for oxidative stress. Intakes of various doses of a mixture of bilberry juice and black tea, rich in flavonoids for four weeks, increased antioxidant capacity in some groups, but urine levels of F2-isoprostanes were not affected. There were substantial individual variations in responses to the drinks related to baseline antioxidant capacity. Supplementation with eicosapentaenoic acid and docosahexaenoic acid decreased the plasma levels of F2-isoprostanes, but not prostaglandin F2α formation or antioxidant capacity. It was concluded that a high intake of foods rich in antioxidants is related to improved antioxidant status. After intake of foods rich in antioxidants, the antioxidant status may increase, but with considerable individual variation in the responses, which warrants further investigation. Lipid peroxidation in vivo is not easily affected by dietary antioxidants in healthy humans. Although n-3 fatty acids are highly unsaturated, they reduce nonenzymatic free radical-catalyzed lipid peroxidation, but not enzymatic lipid peroxidation.

Observational studies have indicated that fruit and vegetables, and dietary antioxidants may play an important role in reducing the risk of chronic diseases, potentially by affecting pathogenic mechanisms such as oxidative stress and inflammation. Clinical trials investigating the effects of supplementation with single or a few antioxidants in high doses have, however, shown inconsistent results and thus have not been able to support the observational findings. It was therefore hypothesised that a supplement, containing a combination of antioxidants mainly extracted from fruit and vegetables, and supplied at moderate doses, might act more beneficially than single antioxidants given at pharmacological doses. The effects of such a supplement were investigated in two interventional studies described in this thesis. The effects on antioxidant status, metabolic control, oxidative stress and inflammation were investigated in overweight men and in patients with type 2 diabetes, subjects that could be expected to have elevated levels of oxidative stress and inflammatory activity. The results of the studies did not support the hypothesis that supplementation with antioxidants from fruit and vegetables may have beneficial effects by counteracting oxidative stress and inflammation, despite markedly increased plasma antioxidant concentrations. However, interesting associations were observed in diabetes patients at baseline between intake of antioxidant rich food as well as levels of antioxidants in plasma, and markers of oxidative stress and inflammation. These associations are compatible with the hypothesis that a high intake of fruit and vegetables and dietary antioxidants decrease oxidative stress levels, have anti-inflammatory effects and a beneficial influence on glycaemic control. The results also indicated that glycaemic control may affect the level of oxidative stress. The absence of beneficial effects from antioxidants might to some extent be explained by the initial levels of oxidative stress and inflammation and by the antioxidative status in the subjects included in the studies. Since the levels generally were comparable with those observed in healthy subjects, this might have decreased the ability to observe any beneficial effects of supplementation with additional antioxidants. Continued investigations are needed to characterise the individuals who potentially might benefit from antioxidant supplementation. In view of apparent positive effects from a high intake of fruit and vegetables found in observational studies and until more knowledge is available from interventional trials about possible benefits and potential risks of antioxidant supplementation it still seems reasonable to recommend a diet rich in fruit and vegetables.