Manufacturers of amino acid supplements claim that they can act as natural stercoids. Eighteen experienced male weight lifters were studied to test this hypothesis for the amino acid arginine. / Master of Science

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The flux of free and peptide amino acids across the stomach and intestinal tissues was quantified using sheep and calves maintained in "steady state” conditions by feeding at hourly intervals. Crossbred wethers and Holstein steers were surgically cannulated in the abdominal aorta, mesenteric vein and portal vein. All animals were fed an orchardgrass, corn, SBM-based diet. The steers received three abomasal infusions; a control solution, and an amino acid mixture simulating casein and casein each at a rate equivalent to 25% of daily crude protein intake. Nutrient fluxes from the mesenteric and portal-drained viscera were measured; non-mesenteric flux was calculated as the difference between portal flux and mesenteric flux. Results of this study support the concept that free amino acids are absorbed by the small intestine and not by the stomach. The flux of peptide amino acids across the portal-drained-viscera indicate that a major portion of the amino acids which are absorbed by cattle and sheep are absorbed in the form of peptides from the stomach. The observation that large quantities of peptide amino acids are absorbed from the stomach is unique and it is expected that this most important discovery will revolutionize the feeding of ruminants. / Ph. D.

Twelve male weight lifters were used to study the effect of oral arginine consumption on growth hormone (hGH) , arginine (Arg), and somatomedin (IGF-1) concentrations. Subjects were randomly assigned to treatment or placebo groups. Following a week of controlled exercise and diet, fasted subjects took either an acute dose of arginine hydrochloride or casein (placebo), at 0.1 g/kg BW. Blood samples were drawn every 15 min, for 90 min after ingestion. All samples were analyzed for hGH and Arg, and IGF-1 was measured in the baseline sample. Subjects then underwent 10 days of caloric restriction, consuming a liquid diet of 22 kcal/kg BW/d. Subjects took either arginine or casein supplements in two doses of 0.1 g/kg BW twice daily, for a total dose of 0.2 g/kg BW/d. The weight lifting protocol remained unchanged. On day 17, subjects were again tested for the acute response to a single dose of 0.1 g/kg BW supplement (arginine or placebo) over 90 min. Blood was collected and analyzed as before. No change in hGH and Arg occurred 90 min after consumption of the arginine supplement, regardless of dietary condition. However, arginine supplementation caused overall higher hGH and Arg levels (2.64 ng/ml and 0.87 mmol/l) compared to placebo (1.36 ng/ml and 0.71 mmol/l) during caloric restriction. IGF-1 did not change in either group under either diet. Thus, oral arginine supplementation at 0.1 g/kg BW did not produce a rise in hGH or Arg after 90 min, and did not affect overall hGH status, as measured by IGF-1, during energy restriction and supplementation. / Master of Science

Methionine- and lysine-containing di- to octapeptides were evaluated for their ability to serve as methionine and lysine sources respectively for the synthesis of secreted proteins. Mammary tissue explants from lactating (10 to 11 d) CD-1 mice and cultured bovine mammary epithelial cells (MAC-T) were used as experimental models. Explants and cultured cells were incubated at 37°C in a humidified atmosphere of 90% air/10% CO₂ and 95% air/5% CO₂, respectively, for 1 to 24 h in Dulbecco's modified Eagle's medium containing hormones, ³H-leucine, and methionine or lysine substrate in either free or peptide-bound form. The ability of methionine and lysine substrates to promote incorporation of ³H-leucine into secreted proteins was quantified. Mouse mammary explants were able to utilize methionine and lysine from all peptides tested except the lysyl octapeptide. All the methionyl peptides were at least as effective as free methionine in promoting ³H-leucine incorporation into secreted proteins. Most methionyl di- and tri-peptides promoted 15 to 76% greater (P < .05) ³H-leucine incorporation than did free methionine. A negative correlation (r = -.89, P < .01) was detected between the rate of ³H-leucine incorporation and the number of amino acid residues in the peptides. The incorporation of ³H-leucine promoted by some methiony] dipeptides was reduced (P < .05) in the presence of a 200-fold higher concentration of glycylsarcosine or carnosine. Incorporation of ³H-leucine promoted by lysyl peptides ranged from 91 to 117% of the incorporation promoted by free lysine. MAC-T cells were also able to utilize methionine from all di- and tri-peptides studied. The ability of the peptides to promote ³H-leucine incorporation varied with experimental conditions. For cells allowed to grow/differentiate for 3 or 8 d, incorporation of ³H-leucine promoted by peptides ranged from 67 to 85% and 86 to 110% of the incorporation promoted by free methionine, respectively. The effect of extracellular matrix on the utilization of peptide-bound methionine by MAC-T cells was also examined. Generally, there was no difference in ³H-leucine incorporation/DNA promoted by methionyl dipeptides in MAC-T cells cultured on matrigel, collagen, laminin, or fibronectin coated or uncoated plates. These results suggest that peptides can serve as sources of amino acids for the synthesis of secreted proteins by both lactating mammary explants and cultured mammary epithelial cells. Mouse mammary explants appear to have a greater ability to utilize peptide-bound methionine than to utilize peptide-bound lysine. Mediated transport of some methiony] peptides may be involved in the peptide utilization by the mammary explants. More extensive utilization of peptides by MAC-T cells following a longer (3 vs 8 d) growth/differentiation period may indicate that some maturation process(s) in the cells was necessary for the most effective utilization of peptides. / Ph. D.

Peroxinitrito (ONOO- + ONOOH), o produto da rápida reação do óxido nítrico com o ânion radical superóxido, tem recebido muita atenção como possível mediador dos efeitos deletérios associados a uma superprodução de &#8226;NO. O peroxinitrito é um potente oxidante que é capaz de oxidar e nitrar várias biomoléculas por mecanismos que contribuímos para esclarecer no decorrer desta tese. Especificamente, estudamos a oxidação de urato e tirosina por peroxinitrito. Demonstramos que o urato é oxidado por peroxinitrito a alantoina, aloxana e ao radical aminocarbonila. Como a reação direta entre urato e peroxinitrito tem uma constante de velocidade relativamente baixa (k= 4,8 x 102 M -1.s-1) em comparação com àquelas de outras biomoléculas, sugerimos que o urato é um potente sequestrador dos radicais derivados do peroxinitrito (&#8226;NO2 e CO3&#8226;-, na maioria dos ambientes biológicos; a pH ácido, o radical &#8226;OH também pode se tornar relevante). No caso da tirosina, confirmamos que ela não reage diretamente com o peroxinitrito mas com os radicais dele derivados. Como antecipado, o rendimento relativo dos produtos (3-nitrotirosina, 3,3-bitirosina e 3-hidroxitirosina (DOPA)) variou com o pH e a presença de CO2. Esses estudos nos levaram a propor a co-localização de proteínas nitradas e hidroxiladas como um possível biomarcador de peroxinitrito. Para testar essa hipótese, um anticorpo monoclonal anti-DOPA foi desenvolvido e utilizado em modelos de infecção por Leishmania amazonenses (macrófagos (J774), e camundongos resistentes (C56Bl/6) e suscetíveis (BALB/c). A co-localização de proteínas hidroxiladas e nitradas ficou evidênciada em todos os modelos testados e ocorreu concomitantemente a máxima produção de &#8226;NO. Infelizmente, o anticorpo obtido perdeu a atividade e ainda não pudemos confirmar esses dados. / Peroxynitrite (ONOO- + ONOOH), which is formed by the fast reaction between nitric oxide and superoxide anion, has been receiving increasing attention as a mediator of the deleterious effects associated with an overproduction of &#8226;NO. The compound is a strong oxidant that is able to oxidize and nitrate a variety of biotargets by mechanisms that this work has contributed to establish. Specifically, we studied the oxidation of urate and tyrosine by peroxynitrite. Urate oxidation produced allantoin, alloxan and the amiocarbonyl radical. Since the rate constant of the direct reaction between urate and peroxynitrite (k= 4,8 x 102 M-1.s-1) is low in comparison with those of other biotargets, we proposed that urate is an efficient scavenger of peroxynitrite-derived radicals (&#8226;NO2 and CO3&#8226;- in most biological environments; at acid pH, the &#8226;OH radical may also become relevant). ln the case of tyrosine, we confirmed that it does not react directly with peroxynitrite but, instead, with the radicals derived form it. As anticipated, the relative yield of the products (3-nitrotyrosine, 3,3-bityrosine and 3-hydroxytyrosine (DOPA)) varied with the pH and CO2 presence. These results led us to propose that co-localization of nitrated and hydroxylated proteins could be a peroxynitrite biomarker. To test this hypothesis, a monoclonal anti-DOPA antibody was developed and tested in Leishamnia amazonensis infection models (macrophages (J774), and resistant (C56Bl/6) and susceptible mice (BALB/c). It was possible to evidence co-localization of hydroxylated and nitrated proteins in all tested models in a time when &#8226;NO synthesis was maximum. Unfortunetly, we were unable to confirm these results due to antibody inactvation; new antibody baches are being obtained.

Peroxinitrito (ONOO- + ONOOH), o produto da rápida reação do óxido nítrico com o ânion radical superóxido, tem recebido muita atenção como possível mediador dos efeitos deletérios associados a uma superprodução de &#8226;NO. O peroxinitrito é um potente oxidante que é capaz de oxidar e nitrar várias biomoléculas por mecanismos que contribuímos para esclarecer no decorrer desta tese. Especificamente, estudamos a oxidação de urato e tirosina por peroxinitrito. Demonstramos que o urato é oxidado por peroxinitrito a alantoina, aloxana e ao radical aminocarbonila. Como a reação direta entre urato e peroxinitrito tem uma constante de velocidade relativamente baixa (k= 4,8 x 102 M -1.s-1) em comparação com àquelas de outras biomoléculas, sugerimos que o urato é um potente sequestrador dos radicais derivados do peroxinitrito (&#8226;NO2 e CO3&#8226;-, na maioria dos ambientes biológicos; a pH ácido, o radical &#8226;OH também pode se tornar relevante). No caso da tirosina, confirmamos que ela não reage diretamente com o peroxinitrito mas com os radicais dele derivados. Como antecipado, o rendimento relativo dos produtos (3-nitrotirosina, 3,3-bitirosina e 3-hidroxitirosina (DOPA)) variou com o pH e a presença de CO2. Esses estudos nos levaram a propor a co-localização de proteínas nitradas e hidroxiladas como um possível biomarcador de peroxinitrito. Para testar essa hipótese, um anticorpo monoclonal anti-DOPA foi desenvolvido e utilizado em modelos de infecção por Leishmania amazonenses (macrófagos (J774), e camundongos resistentes (C56Bl/6) e suscetíveis (BALB/c). A co-localização de proteínas hidroxiladas e nitradas ficou evidênciada em todos os modelos testados e ocorreu concomitantemente a máxima produção de &#8226;NO. Infelizmente, o anticorpo obtido perdeu a atividade e ainda não pudemos confirmar esses dados. / Peroxynitrite (ONOO- + ONOOH), which is formed by the fast reaction between nitric oxide and superoxide anion, has been receiving increasing attention as a mediator of the deleterious effects associated with an overproduction of &#8226;NO. The compound is a strong oxidant that is able to oxidize and nitrate a variety of biotargets by mechanisms that this work has contributed to establish. Specifically, we studied the oxidation of urate and tyrosine by peroxynitrite. Urate oxidation produced allantoin, alloxan and the amiocarbonyl radical. Since the rate constant of the direct reaction between urate and peroxynitrite (k= 4,8 x 102 M-1.s-1) is low in comparison with those of other biotargets, we proposed that urate is an efficient scavenger of peroxynitrite-derived radicals (&#8226;NO2 and CO3&#8226;- in most biological environments; at acid pH, the &#8226;OH radical may also become relevant). ln the case of tyrosine, we confirmed that it does not react directly with peroxynitrite but, instead, with the radicals derived form it. As anticipated, the relative yield of the products (3-nitrotyrosine, 3,3-bityrosine and 3-hydroxytyrosine (DOPA)) varied with the pH and CO2 presence. These results led us to propose that co-localization of nitrated and hydroxylated proteins could be a peroxynitrite biomarker. To test this hypothesis, a monoclonal anti-DOPA antibody was developed and tested in Leishamnia amazonensis infection models (macrophages (J774), and resistant (C56Bl/6) and susceptible mice (BALB/c). It was possible to evidence co-localization of hydroxylated and nitrated proteins in all tested models in a time when &#8226;NO synthesis was maximum. Unfortunetly, we were unable to confirm these results due to antibody inactvation; new antibody baches are being obtained.

Nível elevado de homocisteína (Hcy) no plasma é considerado fator de risco de doença cardiovascular. Consumo reduzido de vitaminas B6, B12 e ácido fólico tem sido relacionado com hiperhomocisteinemia. O objetivo desse estudo foi verificar o consumo de vitaminas B6, B12 e ácido fólico nas populações urbana e rural, bem como a correlação dos níveis plasmáticos dessas vitaminas com os níveis plasmáticos de Hcy. Também determinamos os níveis séricos de lipídeos e avaliamos o risco cardiovascular das populações frente a hiperlipemia. O consumo de B6 e ácido fólico é maior na população urbana, com p=0,00 e p=0,04 respectivamente, sendo o consumo de B12 maior na população rural, com p=0,47. As correlações são significativamente negativa entre Hcy e as vitaminas B12 e ácido fólico . A população rural apresenta Hcy com valor médio de 16,5±9,2&#181;mol/L, classificada como hiperhomocisteinemia moderada, e a população urbana 12,8±5,5 &#181;mol/L, o qual está dentro da faixa de referência. O valor médio de LDL sérica é maior na população urbana (3,4±0,8mmoI/L) do que na população rural (2,8±0,9mmoI/L), com valor de p=0,00. Como fator de risco cardiovascular, consideramos Hcy plasmática >14&#181;mol/L e LDL sérica >3,38mmol/L. Neste caso, 41,4% da população rural e 7,4% população urbana apresentam Hcy maior que 14&#181;mol/L. O inverso ocorre em relação a LDL, onde 43,2% da população urbana e 11% na população rural apresentam níveis acima de 3,38mmol/L. Concluímos que o risco cardiovascular decorrente de hiperhomocisteinemia é maior na população rural que na urbana e este risco poderia reduzir mediante o consumo de vitaminas. / Elevated levels of plasma homocysteine (Hey) are considered a risk factor for cardiovascular diseases. Low intake of vitamins 86, 812 and folic acid have been related to hyperhomocysteinemia. The purpose of the present study is to determine the consumption of the vitamins B6, B12 and folic acid in two Brazilian urban and rural populations, along with the plasmatic levels of these vitamins and plasmatic homocysteine. In addition, the serum levels of lipids have been determined to evaluate the cardiovascular risk in the two populations regarding their hyperlipidemie comdition. The consumption of B6 and folic acid is higher in the urban population (p=0.00 and p=0.04 respective/y), while the consumption of B12 is not significantly different (p=0.47). There is a negative correlation between B12 and folic acid with Hcy. The rural population shows mean Hcy value of 16.5±9.2&#181;mol/L and is classified as having moderate hyperhomocysteinemia, while for the urban population, the mean value is 12.8±5.5&#181;mol/L and is well within the normal range. The mean value of the serum LDL is higher in the urban population (3.4±0.8mmol/L) compared to the rural population (2.8±0.9mmol/lL) with a significance of p=0.00. Plasma Hcy values >14&#181;mol/L and serum LDL >3.38mmol/L were considered as the risk factors for cardiovascular disease. With in the reference values, 41.4% of the rural population and 7.4% of the urban population showa Hcy as a risk factor. For LDL, the inverse is true, i.e 43.2% of urban and 11% of the rural population are at risk. We conclude that the cardiovascular risk arising from hyperhomocysteinemia is higher in the rural population and that this can be reduced by increased consumption of vitamins.

O Trypanosoma cruzi é capaz de matabolizar aminoácidos como fontes de carbono e energia, dentre eles, o aminoácido histidina (His). Canonicamente, a via de degradação de His compreende quatro passos enzimáticos que consistem na oxidação de His a glutamato (Glu). O Glu gerado é convertido em alfa-cetoglutarato (&#945;-KG), que é incorporado ao ciclo de Krebs (TCA), gerando coenzimas reduzidas que irão fornecer elétrons para a cadeia transportadora de elétrons (CTE), produzindo ATP. Nesse trabalho foi possível demonstrar que o substrato da terceira enzima envolvida na degradação de His (imidazolona propionase-TcIP), chamado 4-imidazolona-5-propionato (IPA) é oxidado não enzimáticamente a &#945;-KG, e este é capaz de ser metabolizado diretamente via TCA. Ainda foi possível demonstrar que essa enzima se encontra formando um complexo macromolecular com a segunda enzima da via (urocanato hidratase TcUH). Finalmente, obervou-se que o controle da expressão da TcIP é importante no processo de metaciclogênse de T. cruzi. / Trypanosoma cruzi is able to catabolize amino acids as carbon and energy sources, among them the amino acid histidine (His). Canonically, the His degradation pathway comprises four enzymatic steps consisting of the oxidation of His to glutamate (Glu). The Glu is converted to alpha ketoglutarate (&#945;-KG), which is incorporated into the Krebs cycle (TCA), generating reduced coenzymes that will supply electrons to the electron transport chain (ETC), producing ATP. In this work it was possible to demonstrate that the substrate of the third enzyme involved in the His degradation (imidazolonepropionase - TcIP), called 4-imidazolone-5-propionate (IPA), is non-enzymatically oxidized to &#945;-KG, which is able to be metabolized directly through TCA. It was possible to demonstrate that this enzyme is forming a macromolecular complex with the second enzyme (urocanate hydratase - TcUH). Finally, it was established that the control of TcIP expression is important in the metacyclogenesis process in T. cruzi.

Nível elevado de homocisteína (Hcy) no plasma é considerado fator de risco de doença cardiovascular. Consumo reduzido de vitaminas B6, B12 e ácido fólico tem sido relacionado com hiperhomocisteinemia. O objetivo desse estudo foi verificar o consumo de vitaminas B6, B12 e ácido fólico nas populações urbana e rural, bem como a correlação dos níveis plasmáticos dessas vitaminas com os níveis plasmáticos de Hcy. Também determinamos os níveis séricos de lipídeos e avaliamos o risco cardiovascular das populações frente a hiperlipemia. O consumo de B6 e ácido fólico é maior na população urbana, com p=0,00 e p=0,04 respectivamente, sendo o consumo de B12 maior na população rural, com p=0,47. As correlações são significativamente negativa entre Hcy e as vitaminas B12 e ácido fólico . A população rural apresenta Hcy com valor médio de 16,5±9,2&#181;mol/L, classificada como hiperhomocisteinemia moderada, e a população urbana 12,8±5,5 &#181;mol/L, o qual está dentro da faixa de referência. O valor médio de LDL sérica é maior na população urbana (3,4±0,8mmoI/L) do que na população rural (2,8±0,9mmoI/L), com valor de p=0,00. Como fator de risco cardiovascular, consideramos Hcy plasmática >14&#181;mol/L e LDL sérica >3,38mmol/L. Neste caso, 41,4% da população rural e 7,4% população urbana apresentam Hcy maior que 14&#181;mol/L. O inverso ocorre em relação a LDL, onde 43,2% da população urbana e 11% na população rural apresentam níveis acima de 3,38mmol/L. Concluímos que o risco cardiovascular decorrente de hiperhomocisteinemia é maior na população rural que na urbana e este risco poderia reduzir mediante o consumo de vitaminas. / Elevated levels of plasma homocysteine (Hey) are considered a risk factor for cardiovascular diseases. Low intake of vitamins 86, 812 and folic acid have been related to hyperhomocysteinemia. The purpose of the present study is to determine the consumption of the vitamins B6, B12 and folic acid in two Brazilian urban and rural populations, along with the plasmatic levels of these vitamins and plasmatic homocysteine. In addition, the serum levels of lipids have been determined to evaluate the cardiovascular risk in the two populations regarding their hyperlipidemie comdition. The consumption of B6 and folic acid is higher in the urban population (p=0.00 and p=0.04 respective/y), while the consumption of B12 is not significantly different (p=0.47). There is a negative correlation between B12 and folic acid with Hcy. The rural population shows mean Hcy value of 16.5±9.2&#181;mol/L and is classified as having moderate hyperhomocysteinemia, while for the urban population, the mean value is 12.8±5.5&#181;mol/L and is well within the normal range. The mean value of the serum LDL is higher in the urban population (3.4±0.8mmol/L) compared to the rural population (2.8±0.9mmol/lL) with a significance of p=0.00. Plasma Hcy values >14&#181;mol/L and serum LDL >3.38mmol/L were considered as the risk factors for cardiovascular disease. With in the reference values, 41.4% of the rural population and 7.4% of the urban population showa Hcy as a risk factor. For LDL, the inverse is true, i.e 43.2% of urban and 11% of the rural population are at risk. We conclude that the cardiovascular risk arising from hyperhomocysteinemia is higher in the rural population and that this can be reduced by increased consumption of vitamins.