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<p> Exercise intensity is one of the major factors determining the utilization of carbohydrates (CHO) and lipids in mammalian skeletal muscle. Using indirect calorimetry, we determined maximal oxygen uptake (VO2max) and whole-body rates of CHO and lipid oxidation in rats selectively bred for high and low running capacity (HCR's and LCR's) during exercise at 50, 60, 70 and 80%VO2max. Previous studies have revealed a pattern of selection where mammals with different aerobic capacities use the same proportions of lipids and CHO when exercising at the same relative exercise intensity and as intensity increases, CHO use increases and lipid use decreases. The present results showed that the HCR's had a VO2max and distance run to exhaustion that was 1.3 and 4.0 times greater than the LCR's respectively. Also, both groups of rats followed the pattern of fuel selection seen in previous studies where the same proportions (in%) of lipids and CHO are used at the same relative exercise intensity. On an absolute scale, the HCR's used more lipids and CHO than the LCR's at all exercise intensities but the results were not always statistically significant. We also determined the exercise intensity that elicited the greatest lipid use to be 60% VO2max in both groups.</p> <p> In order to explain these patterns of fuel selection, metabolic indicators, metabolites and enzymes, in skeletal muscle were measured at rest and post exercise for one hour at 60%VO2max. Specifically, ATP and phosphocreatine (PCr) metabolite concentrations were determined in the medial and lateral gastrocnemius, extensor digitorum longus (EDL), tibialis anterior (TA), and soleus muscle. The medial gastrocnemius and soleus were analyzed (pre and post exercise samples were combined) for their oxidative and glycolytic enzyme activity by measuring citrate synthase (CS), cytochrome oxidase (COX), β-hydroxyacyl CoA dehydrogenase (HOAD), and lactate dehydrogenase (LDH) . PCr and ATP concentrations did not change pre and post exercise and between the HCR's and LCR's except for the EDL where there was a significant decrease (P<0.05) in both metabolites after exercise in both groups of rats. For the enzyme measurements, CS and COX activities were higher (P<0.05) in the HCR's for the soleus and HOAD activities were also higher in the HCR's medial gastrocnemius compared to the LCR's. We concluded that the HCR's have a greater oxidative capacity as shown by their greater aerobic and endurance capacity (VO2max and distance to exhaustion), their ability to oxidize a greater absolute amount of lipids and CHO's at the same relative exercise intensity, and their higher activities of oxidative enzymes in the soleus (CS and COX) and medial gastrocnemius (HOAD). Future research into the mechanisms involved in explaining these patterns of fuel selection may include examining fatty acid transport proteins, fatty acid and CHO availability, fiber types, and catecholamines.</p> / Thesis / Master of Science (MSc)

Doctor of Philosophy(PhD) / Atherosclerosis manifests a state of increased oxidative stress characterized by comparable lipid and protein oxidation in the affected arterial wall. While oxidative modification of low density lipoprotein (LDL) has been extensively studied, increasing attention has been focused recently on oxidation of high-density lipoproteins (HDL) and its functional consequences in relation to atherosclerosis. Oxidative modification is thought to generate “dysfunctional” HDL that has lost anti-atherosclerotic activities, including the ability to remove cholesterol from lipid-laden cells. Therefore, there has been much interest in the detection of oxidized HDL. Unfortunately, available methods to detect oxidized HDL are limited at present, in part because oxidative modification of HDL is a complex process and ‘oxidized HDL’ is not a chemically defined entity. What is known however is that conversion of methionine (Met) residues of apolipoprotein (apo) A-I to methionine sulfoxide (MetO) is a process that occurs commonly as HDL undergoes oxidative modification. For example, human apoA-I+16 (containing MetO86 or MetO112) and apoA-I+32 (MetO86 plus MetO112) are generated when apoA-I reacts with lipid hydroperoxides formed as a consequence of the lipoprotein being exposed to 1e−oxidants. The formation of MetO in apoA−I induced by 2e−oxidants (i.e., hydrogen peroxide, hypochlorous acid or myeloperoxidase/hydrogen peroxide/chloride system) is associated with an impaired ability of the apolipoprotein to facilitate reactions relevant to reverse cholesterol transport. In addition, a previous study has suggested the plasma content of apoA-I+32 to be increased in certain subjects that have an increased risk to develop cardiovascular disease (CVD). Moreover, the MetO content in circulating, HDL−associated apoA−I is elevated in type 1 diabetes, a disorder commonly associated with increased oxidative stress and a risk factor for atherosclerosis. Therefore, in the present study, an existing HPLC method was applied to HDL samples from the Fletcher−Challenge study, a nested case control study, to test the potential usefulness of MetO-containing apoA-I as a marker of oxidative stress and/or CVD in a general population. Plasma samples whose HDL contained detectable apoA-I+16 and/or apoA-I+32 had significantly elevated levels of F2-isoprostanes, a marker of in vivo lipid oxidation, consistent with MetO-containing apoA-I being a useful marker of in vivo protein oxidation. Despite this however, there was no significant difference between controls and cases in their concentrations of HDL apoA-I+16 and apoA-I+32 or F2-isoprostanes, suggesting that markers of protein and lipid oxidation are not associated with the risk of coronary heart disease (CHD) in this general population. A limitation of the Fletcher−Challenge study was that only 22% of the 534 HDL samples analyzed contained apoA-I+16 and/or apoA-I+32. In addition, the HPLC−based method used is expensive and time−consuming and may lack the sensitivity needed for apolipoproteins to clinical studies. Thus, a mouse monoclonal anti-human apoA-I+32 antibody (MOA−1) was raised using HPLC−purified apoA-I+32 as immunogen. A sensitive ELISA was then developed using a commercial anti-human apoA-I monoclonal antibody as capture and biotinylated MOA−1 as detection antibody, respectively. The assay detected lipid−free HPLC−purified human apoA-I+32 in a concentration-dependent manner and with a significantly lower limit of detection (i.e., 3 ng/mL) than the HPLC method (1 μg/mL). The ELISA also detected lipid-free apoA-I modified by 2e-oxidants (hydrogen peroxide, hypochlorous acid, peroxynitrite), and HDL oxidized by 1e- or 2e-oxidants and present in buffer or human plasma. Moreover, the extent of recognition of MetO by MOA−1 increased with increasing numbers of MetO in apoA−I, as assessed by the experiments with H2O2−oxidized forms of apoA−I mutants, in which one, two or three Met residues were replaced with Leu. Their detection was concentration-dependent, reproducible, and exhibited a linear response over a physiologically plausible range of concentrations of oxidized HDL. In contrast, MOA-I failed to recognize native apoA-I, native apoA-II, apoA-I modified by hydroxyl radicals or metal ions, or LDL modified by 2e-oxidants. Furthermore, MOA−1 did not detect other Met−containing proteins oxidized by either hypochlorous acid or hydrogen peroxide. Taken together, the results showed that recognition of oxidized proteins by MOA−1 is limited to MetO contained in apoA−I. Finally, in a pilot study, plasma samples obtained from subjects with coronary artery disease (CAD) proven by angiography, and samples from CAD patients undergoing percutaneous coronary intervention (PCI) were analyzed by the ELISA. The preliminary data obtained showed elevated levels of MetO-containing apoA-I in plasma samples of CAD patients compared to those of corresponding control subjects. Unexpectedly, levels of MetOcontaining apoA-I decreased PCI compared to before PCI. A possible explanation for these results is that HDL−associated apoA−I become displaced by acute phase proteins, such as serum amyloid A, in response to PCI. In summary, the ELISA developed here specifically detects apoA-I containing MetO in HDL and human plasma. As such it may provide a useful tool for investigating the relationship between oxidized HDL and CAD.

Doctor of Philosophy(PhD) / Atherosclerosis manifests a state of increased oxidative stress characterized by comparable lipid and protein oxidation in the affected arterial wall. While oxidative modification of low density lipoprotein (LDL) has been extensively studied, increasing attention has been focused recently on oxidation of high-density lipoproteins (HDL) and its functional consequences in relation to atherosclerosis. Oxidative modification is thought to generate “dysfunctional” HDL that has lost anti-atherosclerotic activities, including the ability to remove cholesterol from lipid-laden cells. Therefore, there has been much interest in the detection of oxidized HDL. Unfortunately, available methods to detect oxidized HDL are limited at present, in part because oxidative modification of HDL is a complex process and ‘oxidized HDL’ is not a chemically defined entity. What is known however is that conversion of methionine (Met) residues of apolipoprotein (apo) A-I to methionine sulfoxide (MetO) is a process that occurs commonly as HDL undergoes oxidative modification. For example, human apoA-I+16 (containing MetO86 or MetO112) and apoA-I+32 (MetO86 plus MetO112) are generated when apoA-I reacts with lipid hydroperoxides formed as a consequence of the lipoprotein being exposed to 1e−oxidants. The formation of MetO in apoA−I induced by 2e−oxidants (i.e., hydrogen peroxide, hypochlorous acid or myeloperoxidase/hydrogen peroxide/chloride system) is associated with an impaired ability of the apolipoprotein to facilitate reactions relevant to reverse cholesterol transport. In addition, a previous study has suggested the plasma content of apoA-I+32 to be increased in certain subjects that have an increased risk to develop cardiovascular disease (CVD). Moreover, the MetO content in circulating, HDL−associated apoA−I is elevated in type 1 diabetes, a disorder commonly associated with increased oxidative stress and a risk factor for atherosclerosis. Therefore, in the present study, an existing HPLC method was applied to HDL samples from the Fletcher−Challenge study, a nested case control study, to test the potential usefulness of MetO-containing apoA-I as a marker of oxidative stress and/or CVD in a general population. Plasma samples whose HDL contained detectable apoA-I+16 and/or apoA-I+32 had significantly elevated levels of F2-isoprostanes, a marker of in vivo lipid oxidation, consistent with MetO-containing apoA-I being a useful marker of in vivo protein oxidation. Despite this however, there was no significant difference between controls and cases in their concentrations of HDL apoA-I+16 and apoA-I+32 or F2-isoprostanes, suggesting that markers of protein and lipid oxidation are not associated with the risk of coronary heart disease (CHD) in this general population. A limitation of the Fletcher−Challenge study was that only 22% of the 534 HDL samples analyzed contained apoA-I+16 and/or apoA-I+32. In addition, the HPLC−based method used is expensive and time−consuming and may lack the sensitivity needed for apolipoproteins to clinical studies. Thus, a mouse monoclonal anti-human apoA-I+32 antibody (MOA−1) was raised using HPLC−purified apoA-I+32 as immunogen. A sensitive ELISA was then developed using a commercial anti-human apoA-I monoclonal antibody as capture and biotinylated MOA−1 as detection antibody, respectively. The assay detected lipid−free HPLC−purified human apoA-I+32 in a concentration-dependent manner and with a significantly lower limit of detection (i.e., 3 ng/mL) than the HPLC method (1 μg/mL). The ELISA also detected lipid-free apoA-I modified by 2e-oxidants (hydrogen peroxide, hypochlorous acid, peroxynitrite), and HDL oxidized by 1e- or 2e-oxidants and present in buffer or human plasma. Moreover, the extent of recognition of MetO by MOA−1 increased with increasing numbers of MetO in apoA−I, as assessed by the experiments with H2O2−oxidized forms of apoA−I mutants, in which one, two or three Met residues were replaced with Leu. Their detection was concentration-dependent, reproducible, and exhibited a linear response over a physiologically plausible range of concentrations of oxidized HDL. In contrast, MOA-I failed to recognize native apoA-I, native apoA-II, apoA-I modified by hydroxyl radicals or metal ions, or LDL modified by 2e-oxidants. Furthermore, MOA−1 did not detect other Met−containing proteins oxidized by either hypochlorous acid or hydrogen peroxide. Taken together, the results showed that recognition of oxidized proteins by MOA−1 is limited to MetO contained in apoA−I. Finally, in a pilot study, plasma samples obtained from subjects with coronary artery disease (CAD) proven by angiography, and samples from CAD patients undergoing percutaneous coronary intervention (PCI) were analyzed by the ELISA. The preliminary data obtained showed elevated levels of MetO-containing apoA-I in plasma samples of CAD patients compared to those of corresponding control subjects. Unexpectedly, levels of MetOcontaining apoA-I decreased PCI compared to before PCI. A possible explanation for these results is that HDL−associated apoA−I become displaced by acute phase proteins, such as serum amyloid A, in response to PCI. In summary, the ELISA developed here specifically detects apoA-I containing MetO in HDL and human plasma. As such it may provide a useful tool for investigating the relationship between oxidized HDL and CAD.

Made available in DSpace on 2017-05-12T14:47:08Z (GMT). No. of bitstreams: 1 Tabata _Z Bischoff.pdf: 1316294 bytes, checksum: b9584d84274ea8030e2ea272ba9f133e (MD5) Previous issue date: 2015-02-12 / Soy is an important Fabaceae, both for its high nutritional value, as for the commercial production of oil, which is mainly used in human food. The reaction oxidation that takes place in oils and fats is one of the main causes of the deterioration of food, causing thus a decrease of the nutritional quality and the quality of crude oil, which can be remedied or even prevented through the storage appropriate the grain, temperature, relative humidity and the dried optimum of the grain. Given the above, the objective of this study was to evaluate the main changes in the quality the soybean oil crude, present in grain the soybeans, from the storage of grain in the temperature of 30 °C and different relative humidity s (59,6, 67 and 76%). For this purpose, used grain on variety SYN 1059 RR, derived from a grain processing company and producer of seeds of western Paraná. Soybeans grain It was packaged in plastic recipients, within were placed in saturated salt solutions so that the grains reach the desired moisture. The analyzes the dried, lipid, acid value, color, antioxidant capacity, specific extension by absorption in the ultraviolet region were made during storage, because the correlation between these properties indicates the degree of oil oxidation and, consequently, the quality of the grain. A completely randomized design, in a split plot and the results were submitted to analysis of variance and mean comparison test. The storage time caused changes in physical-chemical properties of the grains, therefore, the oil was degraded over time. Another factor that influenced the degradation was the relative humidity because, with the smallest lower relative humidity (56,9%) and most (76,0%) showed the largest degradation. / A soja é uma GR importante, tanto por seu alto valor nutricional quanto para a produção do óleo utilizado na alimentação humana. A reação de oxidação que acontece nos óleos e gorduras é uma das causas principais da deterioração em alimentos, acarretando, deste modo, a diminuição da qualidade nutricional e a qualidade do óleo bruto, que pode ser remediada ou prevenida pelo armazenamento apropriado dos grãos, com temperatura, umidade relativa e teor de água do grão ótimos. Diante do exposto, o objetivo do presente estudo foi avaliar as principais alterações na qualidade do óleo de soja bruto, presente em grãos de soja, a partir do armazenamento dos grãos na temperatura de 30 oC e diferentes umidades relativas (59,6, 67 e 76%). Para tanto, foram utilizados grãos da variedade SYN 1059 RR, oriundos de uma empresa beneficiadora de grãos e produtora de sementes da região oeste do Paraná. Os grãos de soja permaneceram em recipientes de plástico, dentro dos quais foram colocadas soluções saturadas de sais para que os grãos atingissem a umidade desejada. As análises de teor de água, lipídeo, índice de acidez, cor, capacidade antioxidante e extinção específica por absorção na região ultravioleta foram realizadas durante o armazenamento, por 180 dias, pois a correlação destas propriedades indicam o grau de oxidação do óleo e, consequentemente, a qualidade do grão. Utilizou-se delineamento inteiramente casualizado, em esquema de parcela subdividida e os resultados obtidos foram submetidos à análise de variância e teste de comparação de médias. O tempo de armazenamento provocou alterações nas propriedades físico-químicas dos grãos, assim sendo, o óleo foi degradado ao longo do tempo. Outro fator que influenciou na degradação foi a umidade relativa do ar, pois, com a menor umidade relativa (56,9%) e a maior (76,0%) ocorreram as maiores degradações.

Made available in DSpace on 2017-07-10T19:23:54Z (GMT). No. of bitstreams: 1 Tabata _Z Bischoff.pdf: 1316294 bytes, checksum: b9584d84274ea8030e2ea272ba9f133e (MD5) Previous issue date: 2015-02-12 / Soy is an important Fabaceae, both for its high nutritional value, as for the commercial production of oil, which is mainly used in human food. The reaction oxidation that takes place in oils and fats is one of the main causes of the deterioration of food, causing thus a decrease of the nutritional quality and the quality of crude oil, which can be remedied or even prevented through the storage appropriate the grain, temperature, relative humidity and the dried optimum of the grain. Given the above, the objective of this study was to evaluate the main changes in the quality the soybean oil crude, present in grain the soybeans, from the storage of grain in the temperature of 30 °C and different relative humidity s (59,6, 67 and 76%). For this purpose, used grain on variety SYN 1059 RR, derived from a grain processing company and producer of seeds of western Paraná. Soybeans grain It was packaged in plastic recipients, within were placed in saturated salt solutions so that the grains reach the desired moisture. The analyzes the dried, lipid, acid value, color, antioxidant capacity, specific extension by absorption in the ultraviolet region were made during storage, because the correlation between these properties indicates the degree of oil oxidation and, consequently, the quality of the grain. A completely randomized design, in a split plot and the results were submitted to analysis of variance and mean comparison test. The storage time caused changes in physical-chemical properties of the grains, therefore, the oil was degraded over time. Another factor that influenced the degradation was the relative humidity because, with the smallest lower relative humidity (56,9%) and most (76,0%) showed the largest degradation. / A soja é uma GR importante, tanto por seu alto valor nutricional quanto para a produção do óleo utilizado na alimentação humana. A reação de oxidação que acontece nos óleos e gorduras é uma das causas principais da deterioração em alimentos, acarretando, deste modo, a diminuição da qualidade nutricional e a qualidade do óleo bruto, que pode ser remediada ou prevenida pelo armazenamento apropriado dos grãos, com temperatura, umidade relativa e teor de água do grão ótimos. Diante do exposto, o objetivo do presente estudo foi avaliar as principais alterações na qualidade do óleo de soja bruto, presente em grãos de soja, a partir do armazenamento dos grãos na temperatura de 30 oC e diferentes umidades relativas (59,6, 67 e 76%). Para tanto, foram utilizados grãos da variedade SYN 1059 RR, oriundos de uma empresa beneficiadora de grãos e produtora de sementes da região oeste do Paraná. Os grãos de soja permaneceram em recipientes de plástico, dentro dos quais foram colocadas soluções saturadas de sais para que os grãos atingissem a umidade desejada. As análises de teor de água, lipídeo, índice de acidez, cor, capacidade antioxidante e extinção específica por absorção na região ultravioleta foram realizadas durante o armazenamento, por 180 dias, pois a correlação destas propriedades indicam o grau de oxidação do óleo e, consequentemente, a qualidade do grão. Utilizou-se delineamento inteiramente casualizado, em esquema de parcela subdividida e os resultados obtidos foram submetidos à análise de variância e teste de comparação de médias. O tempo de armazenamento provocou alterações nas propriedades físico-químicas dos grãos, assim sendo, o óleo foi degradado ao longo do tempo. Outro fator que influenciou na degradação foi a umidade relativa do ar, pois, com a menor umidade relativa (56,9%) e a maior (76,0%) ocorreram as maiores degradações.