Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Made available in DSpace on 2014-06-11T19:23:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-10-10Bitstream added on 2014-06-13T18:50:44Z : No. of bitstreams: 1 silva_wjm_me_arafcf.pdf: 565516 bytes, checksum: cac81b0a06ae75ac946b2b00d27ba27f (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / Alimentos enriquecidos e medicamentos são importantes meios para o fornecimento de vitaminas para o nosso organismo. É fundamental desenvolver métodos de validação de quantificação dos nutrientes para assegurar a qualidade dos resultados analíticos. O ácido fólico é a vitamina B9. Os folatos são as formas da vitamina naturalmente encontradas nos alimentos e correspondem aos compostos que têm atividade vitamínica similar a do ácido fólico, a forma sintética dos folatos que corresponde a forma encontrada em suplementos vitamínicos, medicamentos e alimentos fortificados, por ser mais bem absorvido pelo organismo. Há evidências da relação entre a deficiência de folatos e o risco de defeitos no desenvolvimento de fetos, durante a gestação, doenças cardíacas, de alguns tumores e mal de Alzheimer's, entre outras patologias. A validação é o estabelecimento de uma evidência documentada que fornece alto grau de segurança de que um processo específico levará consistentemente a um resultado confiável, ou seja, é o ato de provar que o procedimento, processo, equipamento, material, atividade ou sistema realmente leva a resultados confiáveis. A espectroscopia UV-Vis trata-se de uma técnica consolidada, de baixo custo e sensibilidade compatível com muitas das necessidades que surgem no controle de qualidade de produtos farmacêuticos e alimentícios. Foi desenvolvido e validado um método espectrofotométrico de quantificação do ácido fólico / Enriched foods and drugs are important means for supplying vitamins for our organism. It is essential to develop of methods for validation to ensure product quality. Folic acid is vitamin B9. Folates are forms of vitamin naturally found in foods and correspond to compounds that have activity similar to vitamin folic acid that is the synthetic form of folates found in vitamin supplements, fortified foods and drugs, to be better absorbed by the body. There is evidence of the relationship between folate deficiency and risk of defects in developing fetuses during pregnancy, cardiac disease, Alzheimer's disease, and certain types of tumors, among others diseases. Validation is establishing documented evidence that provides a high degree of assurance that a specific process will consistently a reliable result, or is the act of proving that the procedure, process, equipment, material, activity or system leads to reliable results. The spectroscopy UV-Vis is a consolidated technique, with low cost and sensitivity compatible with many of the needs that arise in quality control of pharmaceutical products and food. In this work, it was developed and validated a method for spectrophotometric quantification of folic acid

Made available in DSpace on 2014-06-11T19:33:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-08-28Bitstream added on 2014-06-13T18:07:28Z : No. of bitstreams: 1 guarizafilho_o_dr_arafo.pdf: 1240531 bytes, checksum: c7e1cb0f17d9a4a706f95d4f47284151 (MD5) / O objetivo deste trabalho foi elucidar os mecanismos do ácido fólico em prevenir o aparecimento de fenda palatina teratogenicamente induzida pela dexamatasona em ratos. Para tanto, foram usadas um total de 13 ratas do tipo Holtzman. G1) 3 ratas receberam 3mg/Kg Dex. Intramuscular (i.m.) do 14o ao 17o dia de prenhez. G2) 4 ratas receberam 3mg/Kg Dexa (i.m.) do 14o ao 17o dia de prenhez, e uma dose de 5mg/kg de ácido fólico (via oral) do dia 14o até 17o de prenhez. G3) 3 ratas receberam 3mg/Kg Dexa (i.m.) do 14o ao 17o dia de gravidez e uma dose de 5mg/kg de ácido fólico (via oral) do início até o final da prenhez. G4) 3 ratas receberam 3mg/Kg Dex (i.m.) do 14o ao 17o dia de prenhez e uma dose de 5mg/kg de ácido fólico (via oral) desde 5 dias antes da concepção até o final da gravidez. As cabeças dos fetos foram removidas no 19o dia de prenhez e estudadas. Foi utilizado o teste não paramétrico de Kruskal-Wallis. O resultados mostraram o seguinte: G1) dos 27 fetos, (96,30%) apresentaram fenda total, e apenas 1 apresentou fenda parcial. G2) Dos 23 fetos apenas (8,7%) com fenda total, (43,35%) com ausência de fenda e (47,80%) apresentaram fenda parcial. G3) Observou-se 88,24% apresentaram fenda incompleta, 5,88% apresentaram fenda completa e 5,88% ausência de fenda. G4) dos 31 animais, (54,4%) apresentaram fenda incompleta, e 32,26% apresentaram fenda completa e (12,9%) sem fenda. / The aim of the present study is to determine whether (FA) prevented cortisone-induced cleft palate (CP) in the Holtzman mouse. The animals were divided in 4 groups: G1- 3 pregnant mice had 3mg/Kg of Dex intramuscularly administrated (IA) from the 14th to 17th day of gestation; G2- 4 pregnant mice had 3mg/Kg of Dex IA from the 14th to 17th day of gestation, with a daily dose (dd) of 5mg/kg FA oral administrated (OA) from 14th to 17th day of gestation; G3- 3 pregnant mice had 3mg/Kg of Dex IA from the 14th to 17th day of gestation, with a (dd) of 5mg/kg FA OA from day 0 until 17th day of gestation; G4- 3 pregnant mice had 3mg/Kg of Dex IA from 14th to 17th day of gestation, with a (dd) of 5mg/kg FA OA 5 days before the fertilization until the 17th day of gestation. The fetuses were examined on 19th day of gestation. The data was analyzed by not paired Kruskal-Wallis test, and showed statistical difference among average score values. The results demonstrated that (96,3%) (G1) had total CP, and only 1 partial CP. In the (G2), (8,7%) showed total CP, (47,8%) partial CP and (43,35%) showed no CP. The (G3) showed (5,88%) had total CP, (88,24%) partial CP and (5,88%) had no CP. In the (G4), (54,4%) showed partial CP and (32,26%) total CP. Only (12,9%) had no CP. These results indicated that FA reduces the incidence of Dex-induced cleft palate independently on the period of the administration.

Orientador: José Paschoal Batistuti / Banca: Ossamu Hojo / Banca: João Bosco Faria / Resumo: Alimentos enriquecidos e medicamentos são importantes meios para o fornecimento de vitaminas para o nosso organismo. É fundamental desenvolver métodos de validação de quantificação dos nutrientes para assegurar a qualidade dos resultados analíticos. O ácido fólico é a vitamina B9. Os folatos são as formas da vitamina naturalmente encontradas nos alimentos e correspondem aos compostos que têm atividade vitamínica similar a do ácido fólico, a forma sintética dos folatos que corresponde a forma encontrada em suplementos vitamínicos, medicamentos e alimentos fortificados, por ser mais bem absorvido pelo organismo. Há evidências da relação entre a deficiência de folatos e o risco de defeitos no desenvolvimento de fetos, durante a gestação, doenças cardíacas, de alguns tumores e mal de Alzheimer's, entre outras patologias. A validação é o estabelecimento de uma evidência documentada que fornece alto grau de segurança de que um processo específico levará consistentemente a um resultado confiável, ou seja, é o ato de provar que o procedimento, processo, equipamento, material, atividade ou sistema realmente leva a resultados confiáveis. A espectroscopia UV-Vis trata-se de uma técnica consolidada, de baixo custo e sensibilidade compatível com muitas das necessidades que surgem no controle de qualidade de produtos farmacêuticos e alimentícios. Foi desenvolvido e validado um método espectrofotométrico de quantificação do ácido fólico / Abstract: Enriched foods and drugs are important means for supplying vitamins for our organism. It is essential to develop of methods for validation to ensure product quality. Folic acid is vitamin B9. Folates are forms of vitamin naturally found in foods and correspond to compounds that have activity similar to vitamin folic acid that is the synthetic form of folates found in vitamin supplements, fortified foods and drugs, to be better absorbed by the body. There is evidence of the relationship between folate deficiency and risk of defects in developing fetuses during pregnancy, cardiac disease, Alzheimer's disease, and certain types of tumors, among others diseases. Validation is establishing documented evidence that provides a high degree of assurance that a specific process will consistently a reliable result, or is the act of proving that the procedure, process, equipment, material, activity or system leads to reliable results. The spectroscopy UV-Vis is a consolidated technique, with low cost and sensitivity compatible with many of the needs that arise in quality control of pharmaceutical products and food. In this work, it was developed and validated a method for spectrophotometric quantification of folic acid / Mestre

Folates are water-soluble B vitamins, which maintain DNA stability by regulating nucleotide synthesis and DNA methylation. Folates influence CRC risk and their ability to prevent or promote carcinogenesis may be dependent on several variables here investigated. No in vitro study has yet modelled the physiological folate status that human colon cells are exposed to in vivo. This study evaluates the ability of different forms and concentrations of folate to maintain DNA stability and normal cell function in folate-sufficient and stable human colonocytes and to modify DNA instability and the acquisition of abnormal characteristics in folate-deficient and genomically-unstable colonocytes. Non-malignant human NCM460 colonocytes cultured at physiologically-relevant concentrations of 5-methyl-THF or FA, representing the average deficient (2.5 ng/mL), sufficient (10 ng/mL) or highest post-supplementation (100 ng/mL) folate levels found in human plasma were used in this study as a model of colon-folate interaction. This work established that FA is taken up and/or retained to a lesser extent than 5-methylTHF and is less efficient at maintaining DNA stability and normal cellular characteristics in folate-sufficient and genomically-stable colonocytes at baseline, particularly at deficient and sufficient concentrations in the medium to longer term (14-21 days). During repletion of folate-deficient and genomically-unstable cells, sufficient concentrations of FA do not increase intracellular folate status and worsen the unstable phenotype, by perpetuating DNA instability and enabling the acquisition of a more pro-malignant protein expression. On the contrary, employing 5-methyl-THF sufficiency for repletion positively modifies the abnormal protein profile and morphological features of folate-deficient cells, mitigating potential progression to malignant transformation. When high post-supplementation concentrations are employed, both folate forms increase intracellular folate status, but drive a more promalignant and stress-induced proteome profile and, in the case of 5-methyl-THF, promote abnormal cell morphologies. In conclusion, the folate type, concentration employed, baseline folate status and timing of exposure to folate supplementation are important variables that should be taken into account by future studies evaluating the potential impact of mandatory FA fortification on CRC.

Background: In addition to a diet including fortified dietary staples, the use of prenatal multivitamin supplements among women has been shown, in some cases, to lead to excessive micronutrient intake levels for nutrients such as folic acid (FA). It was therefore hypothesized that prenatal vitamin supplementation, in addition to a standard Canadian diet, would place pregnant Canadians at risk for excessive FA intake. With little available research on the potential negative impact of excess FA intake in pregnancy, it was further proposed that high concentrations of FA may adversely affect placental health and function. Thus, the aim of the current study was three-fold: 1) To determine micronutrient intake in a large Canadian cohort of pregnant women; 2) To determine the extent to which FA intake in this cohort may exceed the tolerable upper intake level (UL) after prenatal supplementation; and 3) To determine the effects of excessive FA exposure on placental health and function in vitro. Methodology: Second trimester 3-day food records of pregnant women (N=216) were analyzed for micronutrient intake using ESHA Food ProcessorTM. Nutrient intake values were compared to established Dietary Reference Intake (DRI) values. In a series of experiments, the effects of exogenous folic acid (2-4000 ng/ml) on placental health and function were examined in two placental cell lines [HTR-8/SVneo (N=3) and BeWo (N=3)], and a human placenta explant model (N=6). Following a 48-hour incubation period, the effects of excessive folic acid exposure on placenta cell proliferation, viability, and apoptosis were determined, along with evaluation of placenta cell function via cell invasion and B-hCG hormone release assays. Results: Through dietary sources alone, most pregnant women studied were consuming adequate levels of most micronutrients. However the majority of examined women (>50%) demonstrated a risk of dietary inadequacy for vitamin D, vitamin E, folate, and iron. In the examined cohort, 83% of study participants reported prenatal supplement usage. In vitro exposure of human placenta cells and explants to excessive FA concentrations resulted in no significant differences in cellular proliferation, apoptosis, invasion, or B-hCG hormone production. However, decreased cell viability was observed in BeWo cells at increased FA concentrations (200-2000 ng/mL). Conclusion: Food sources alone do not appear to provide women in Canada with adequate intake of all micronutrients recommended for a healthy pregnancy. Though a prenatal supplement containing FA may be necessary for most women, current FA levels in many prenatal supplements may lead to excessive FA intake above the established UL. Yet, as measured in this study, high FA concentrations do not seem to adversely affect most primary indicators of placental cell health or function.

Maternal consumption of dietary methyl donors (DMDs) such as folate, methionine, choline, as well as co-factors including zinc, vitamins B2, B6 and B12 can lead to permanent alterations in the DNA and gene expression of the developing fetus. This study aimed to identify patterns of DMD intake during the second trimester of pregnancy and their associations with infant birth weight, small and large for gestational age (SGA and LGA, respectively). From food sources alone, most pregnant women were below the estimated average requirement for dietary folate equivalent (DFE) (69%) and below the adequate intake measure for choline (99%). Zinc seemed to be the most important nutrient for attaining adequate birth weight. DFE and vitamin B12 were positively associated with birth weight. DFE also reduced the risk for SGA whereas choline increased the risk for LGA. Therefore, DMD intake from food sources during pregnancy may be important to ensure optimal infant birth weight.

Folate supplementation at a level of 15 to 35 mg per week is routinely prescribed for many chronic hemodialysis patients in B.C. In recent studies involving these levels of folate supplementation, RBC folate concentrations ranged from near the upper limit of normal to 1.5 times this upper limit. Initially there was research suggesting that high dose folate supplementation impaired zinc absorption but more recent studies refute this hypothesis. A beneficial effect of high dose folate supplementation is lowering of plasma homocysteine levels. This may be desirable since the homocysteinemia observed in chronic renal failure patients may be a factor in their commonly occurring premature vascular disease. The present study addressed folate needs on a nutritional basis but did not investigate folate's effect on homocysteine levels. The study involved chronic hemodialysis patients and was designed to: 1. assess whether patients consuming the Recommended Nutrient Intake for folate, require a folate supplement to maintain normal folate stores; 2. assess whether patients receiving a supplement of 5 mg of folate per day will have RBC folate levels exceeding the upper limit of the normal range; 3. compare serum zinc concentrations (and in some cases hair zinc levels as well) of patients receiving no zinc supplement or a 22.5 mg per day zinc supplement, to each other and to normal values; 4. assess whether a supplement of 5 mg of folate per day is associated with impaired zinc status; 5. in the event that a 5 mg per day folate supplement is associated with impaired zinc status, assess whether a supplement of 22.5 mg of zinc per day is associated with an improvement in zinc status; and 6. determine average daily energy, protein, folate and zinc intakes of patients. A 2x2 factorial quasiexperimental design was employed. The study included 21 clinically stable chronic hemodialysis patients between the ages of 25 and 69, who were receiving folate and/or zinc supplements at certain specific levels. Subjects were entered into treatment groups based on the following folate/zinc supplementation levels: no folate, no zinc; no folate, 22.5 mg zinc/day; 5 mg folate/day, no zinc; 5 mg folate/day, 22.5 mg zinc/day. Folate status was assessed using RBC folate concentration. Serum zinc concentration was measured in all subjects. Hair zinc level was determined in 6 of the zinc-supplemented subjects. A food frequency questionnaire was developed to determine dietary folate and zinc intakes. Subjects kept 3 day food records so average daily energy and protein intakes could be determined. Study results indicated no significant difference in protein intake (g/kg b.w.) or energy intake (expressed as a percent of requirement) among the four treatment groups. Differences in dietary folate intakes among the four treatment groups as well as between zinc-supplemented and non zinc-supplemented subjects, were not significant (p≤0.05). Mean dietary folate intake for all study subjects was 4.2 ug/kg b.w. RBC folate concentration was normal in both treatment groups receiving no supplemental folate. In contrast, the RBC folate concentration for both folate-supplemented groups was approximately 6.5 to 7 times the upper limit of the normal range. The difference between RBC folate concentration for folate supplemented and unsupplemented groups was highly significant (p<0.00001) and remained so when analysis of covariance was done with number of months of folate supplementation as the covariate. RBC folate levels did not differ significantly between zinc-supplemented and unsupplemented groups (p≤0.05). Differences in dietary zinc intakes among the four treatment groups as well as between zinc-supplemented and non zinc-supplemented subjects were not significant (p≤0.05). Mean dietary zinc intake for all study subjects was 9.39 mg/day. Serum zinc levels were below normal in both treatment groups receiving no supplemental zinc. The 22.5 mg zinc, no folate group had a serum zinc concentration near the lower limit of the lower range while that in the 22.5 mg zinc, 5 mg folate group was slightly below normal. When all zinc supplemented subjects were combined, serum zinc concentration was just within the normal range. Hair zinc analysis was conducted in a subgroup of 6 zinc-supplemented subjects and a group of non zinc-supplemented healthy controls. Hair zinc level was significantly higher in the zinc-supplemented subjects than in the controls (p≤O.01). In conclusion, folate supplementation does not appear to be required on a nutritional basis in clinically stable chronic hemodialysis patients not receiving medications known to affect folate status, who are consuming a diet providing a minimum of 1 g of protein per kg b.w. and 4.6 ug of folate per kg b.w. The low serum zinc concentrations observed in both zinc-supplemented and non zinc-supplemented patients may have been due to a shift of zinc from serum to other "zinc pools" in the body as reported in the literature. / Land and Food Systems, Faculty of / Graduate

Canada fortifies certain products with folic acid and has periconceptional supplementation guidelines – policies designed to improve folate status and reduce the incidence of poor birth outcomes. Though optimal folate concentrations have been linked to health benefits, concerns have been raised regarding potential associations with adverse health outcomes. Direct biochemical assessment of the folate status of Canadians based on a nationally representative sample has not been done in more than 40 years. The overall purpose of this research was to investigate the folate status of the Canadian population. All analyses used the nationally representative 2007–2009 Canadian Health Measures Survey (CHMS). Red blood cell (RBC) folate was measured by Immulite 2000 immunoassay. Key findings indicate that folate deficiency (<305 nmol/L) was virtually non-existent in the Canadian population (6–79 years old). Still, one-fifth of women of childbearing age (WCBA; 15–45 years old) had sub-optimal concentrations for the prevention of neural tube defects (<906 nmol/L). Folic acid supplement intake was a primary determinant of WCBA achieving a RBC folate concentration ≥906 nmol/L. A distinct shift towards elevated RBC folate concentrations emerged. Three hypothetical cut-offs (1450 nmol/L, 1800 nmol/L and 2150 nmol/L) were examined to create dialogue since a universal definition of high RBC folate concentration does not exist. Females, participants aged 60¬–79 years, and those who were overweight or obese had the greatest prevalence of having high RBC folate at each cut-off. We conducted the first national-level comparison of RBC folate concentrations between the United States and Canada. Two different folate assay methods – microbiologic assay (NHANES) and Immulite 2000 immunoassay (CHMS) – necessitated the application of a conversion equation. Median Canadian RBC folate concentrations (adjusted to microbiologic assay) were lower than those of Americans but unadjusted Canadian median RBC folate values were higher. Canadian WCBA were less likely than American WCBA to have RBC folate ≥906 nmol/L, though Canadian WCBA with unadjusted RBC folate values were more likely to achieve this cut-off. These results indicate a need for strategies targeting WCBA to improve compliance with folic acid supplement recommendations. The strength and necessity of supplements for the general population should be re-assessed. Further, harmonization of folate measurement procedures in future surveillance efforts would support comparisons and inform policy directions.

A epigenética é uma subárea da Genética, na qual são estudados mecanismos que são essenciais para o adequado desenvolvimento dos mamíferos, sendo que, alterações neste estágio podem levar a vários distúrbios metabólicos, como a obesidade. Atualmente a obesidade é um grave problema de saúde pública mundial, tem origem multifatorial envolvendo tanto fatores ambientais quanto genéticos. Existem alguns genes que estão envolvidos com a obesidade, como por exemplo, o gene da pró-opiomelanocortina (POMC). O objetivo do presente projeto de pesquisa é investigar os padrões de expressão e metilação do gene Pomc na prole de ratas submetidas às dietas deficiente e suplementada com ácido fólico. Os animais utilizados foram ratos Wistar. O estudo envolveu filhotes (n=24) machos e fêmeas que foram desmamados com a mesma dieta de suas respectivas mães, sendo três grupos de tratamento, o grupo controle (2,0 mg de ácido fólico/kg de ração), o grupo deficiente (0,5 mg de ácido fólico/kg de ração) e o grupo suplementado (8 mg de ácido fólico/kg de ração). Foram coletadas amostras do núcleo arqueado do hipotálamo, a partir das quais foram extraídos DNA, RNA e proteínas utilizando kits comerciais seguindo o protocolo do fabricante. Com o DNA foi realizada a análise do padrão de metilação. O mRNA foi utilizado para a análise da expressão gênica, por PCR em tempo real, pelo sistema TaqMan® (Life Technologies(TM)). O estudo de proteômica foi realizado por Western blotting. De modo geral, observou-se que o peso corpóreo dos filhotes machos não apresentou diferença estatística entre os grupos. O consumo de ração do grupo deficiente em ácido fólico foi estatisticamente (p = 0,03) maior do que o grupo controle. Em relação aos filhotes fêmeas observou-se que o peso corpóreo do grupo suplementado foi estatisticamente (p = 0,01) maior do que o grupo controle, e referente ao consumo de ração, não houve diferença estatística significativa entre os grupos de tratamento. As análises de peso cerebral, expressão gênica, metilação e expressão proteica de Pomc não apresentaram diferenças estatísticas significativas entre os grupos de tratamento de ambos os sexos. Conclui-se que a intervenção com dietas com diferentes concentrações de ácido fólico não ocasionou alterações significativas na prole, em relação ao estudo de proteômica e aos padrões de metilação e expressão do gene Pomc. Quanto ao peso corpóreo e consumo de ração dos animais mostrou-se que a suplementação com ácido fólico durante a gestação e no pós desmame foi capaz de alterar estes dois parâmetros, com resposta divergente entre os machos e fêmeas na prole adulta. / Epigenetic mechanisms are essential for proper development in mammals, and that changes at this stage may lead to various metabolic disorders such as obesity. Currently obesity is a serious problem of public health worldwide, has a multifactorial origin involving both environmental and genetic factors. There are some genes that are involved with obesity, such as the proopiomelanocortin gene (POMC). The aim of this research project is to investigate the expression and methylation patterns of the Pomc gene in the offspring of rats subjected to deficient and supplemented diets with folic acid. Animals used were Wistar rats. The study involved males and females pups (n = 24) that were weaned at the same diet their mothers, three treatment groups, control group (2,0 mg/kg of folic acid), deficient group (0,5 mg/kg of folic acid) and the supplemented group (8,0 mg/kg of folic acid). The arcuate nucleus of the hypothalamus tissue were collected, from which was extracted DNA, RNA and proteins using commercially available kits following the manufacturer\'s protocol. The DNA methylation pattern analysis was performed. The mRNA was used for the analysis of gene expression by real time PCR, the TaqMan (Life Technologies (TM)) system. The proteomic study was carried out by Western blotting. In general, we found that the body weight of the male offspring showed no statistical difference between the groups. The feed intake of folic acid deficient group was statistically (p = 0.03) higher than the control group. In relation to female offspring was observed that the body weight of the supplemented group was statistically (p = 0.01) higher than the control group, and related to feed intake, there was no statistically significant difference between the treatment groups. The analysis of brain weight, gene expression, methylation and protein expression of Pomc no significant statistical differences among treatment groups of both sexes. Concluded that the intervention diets with different folic acid concentrations did not cause significant changes in the offspring compared to the study of proteomics and methylation and expression patterns of the Pomc gene. As for the body weight and feed consumption of animals it showed that supplementation with folic acid during pregnancy and post weaning was able to alter these two parameters with differing response between males and females in the adult offspring.