Methylenetetrahydrofolate reductase (MTHFR) catalyses the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a carbon donor for the remethylation of homocysteine to methionine. A common mutation in MTHFR, 677C → T, which converts an alanine (A) to a valine (V) residue, is associated with increased plasma homocysteine and, consequently, is a risk factor for several vasculopathies. Recently, a second common mutation in MTHFR was reported (1298A → C) which converts a glutamate (E) to an alanine (A) residue in the C-terminal regulatory region. Thus study was designed to assess the in vitro and in vivo impact of this polymorphism. Site-directed mutagenesis of the human MTHFR cDNA was performed to create all four combinations of the two common MTHFR polymorphisms. Enzyme activity was assessed in a bacterial expression system. / Since the 1298A → C polymorphism is due to a mutation in the regulatory domain, a parallel study was performed involving the complete deletion of this region. / For in vivo assessment of the 1298A → C variant, we examined this mutation in three clinical populations. / Betaine-homocysteine methyltransferase (BHMT) is a second remethylation enzyme that converts betaine and homocysteine to dimethylglycine and methionine, respectively. This work represents the first report of sequence variants in the newly-cloned BHMT gene. Using SSCP analysis, three common base changes were identified in a panel of healthy controls. (Abstract shortened by UMI.)

Folic acid is a water-soluble, B-complex vitamin influencing a number of biological processes in humans and particularly important in the prevention of neural tube defects (associated with spinal bifida) in unborn children. Reliable analytical methods are therefore needed for quantisation of the amount of total folic acid (FA) in biological materials of quality assurance and regulatory purposes. What is particularly needed are rapid and reliable methods for ensuring that the correct amount of FA is consumed and the degradation rates of these compound is kept at minimum during the extraction process. Analytical methods for determination of folic acid in biological materials have been around for decades and the most common procedures include microbiological assay; biomolecular interaction analysis (BIA); immunoassay; conventional chromatographic procedure such as thin-layer column chromatography (TLC) and high performance liquid chromatography (HPLC). These procedures were replaced by HPLC, which is more rapid and in many instances yields a better resolution. Current HPLC methods uses C-18 column and reverse phase conditions in combination with ion-pair or ion suppression techniques; fluorescence or electrochemical detector, unfortunately, excitation and emission of folic acid is found not sufficiently to allow physiological levels of the form of the vitamin to be detected. In addition, ion-pair reagent nullifies the mobile phase and interferes with the absorption! fluoresce spectrum resulting in poor separation. Therefore this study was carried out to address and improve the problems that are in the existing HPLC methods. Currently scarce information is available on the determination of folic acid in biological materials by HPLC with UV detection. Serum samples were spiked with folic acid standard to check the efficiency of the method. Other wavelengths from 200 nm to 300 nm were attempted for detection of folic acid, in which the wavelength 250 nm was found to have better absorbance compared to other wavelengths. Folic acid was detected at 250 nm wavelength under isocratic elution using a mobile phase consisting of citrate phosphate buffer: acetic acid: methanol. Folic acid in maize meal was detected at 290 nm using mobile phase containing potassium phosphate containing ascorbic acid/sodium ascorbate mixture and 2-mercaptoethanol under gradient elution. The mobile phase used for gradient and isocratic elution was suitable for separation of folic acid from other compounds with flow rate of 3 ml/min modified to Iml/mim to avoid overloading of the column under isocratic elution. For good separation of folic acid under gradient elution the flow rate was set at 0.8ml/min with pH of mobile phase modified from pH 2.2 to pH 2.5. The recovery of folic acid added to human serum was 91% -100% and recovery of folic acid added in unfermented maize meal and fermented maize meal ranged from 55% - 73%. Folic acid level from unfermented maize meal and fermented maize meal ranged between 1.29 - 1.3 [!g/g and 1 - 2.1 [!g/g respectively. In conclusion the optimised method in this study gives better analytical results when compared with earlier HPLC method in terms of efficiency, reproducibility and sensitivity for folic acid in human serum and maize meal. However, there is a need to minimise the loss of folic acid during the sample treatment. The outcome of this work indicated that more work has to be done to improve extraction procedure for specific foods with minimum time preparation to sample analysis. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Durban, 2007.

Folate derivatives are required for nucleotide/DNA synthesis and DNA methylation. Methylenetetrahydrofolate reductase (MTHFR) converts 5,10-methylenctetrahydrofolate to 5-methyltetrahydrofolate, the folate derivative required for homocysteine remethylation to methionine, the precursor of S-adenosylmethionine. Approximately 45%--50% of the general population is heterozygous for a common substitution (677C &rarr; T, A to V) in MTHFR. Due to loss of heterozygosity (LOH) in cancer cells, individuals who are heterozygous for MTHFR in their constitutional DNA may contain only one of the above alleles in their tumor DNA. / Loss of heterozygosity of MTHFR was observed in 40% of ovarian carcinoma tumor samples and in 16% of colon carcinoma samples suggesting that the chromosomal location to which the MTHFR gene maps (1p36.3) undergoes frequent LOH. Examination of cell viability of human fibroblasts and of human colon carcinoma cell lines in minimum essential media (MEM) lacking methionine found both cell types to be extremely sensitive to the methionine deficiency. Replacing methionine with homocysteine and vitamin B12 restored the growth of normal fibroblast lines to levels that approached those of replete MEM, but the transformed lines increased proliferation only slightly under these conditions. These results support earlier reports regarding the increased methionine dependence of transformed lines. Targeting specific MTHFR variants with the antisense oligonucleotide resulted in &sim;50% decreased survival of two carcinoma cell lines (V/V genotype), possibly due to MTHFR's involvement in methionine synthesis. Allele-specific targeting of MTHFR could therefore provide an effective approach for cancer therapy. Furthermore, cancer patients with the V/V genotype may require less aggressive anti-folate chemotherapy since V/V carcinoma lines were highly sensitive to drug treatment (IC50 < 25 nM) whereas the A/A lines were more variable in response.

High vitamin (HV), AIN-93G diet with ten-fold the regular amount of vitamins (RV), consumed by pregnant Wistar rats increases characteristics of metabolic syndrome (MetS) in their first litters. Therefore, the effects of the maternal HV-diet on tissue mechanisms regulating insulin resistance in offspring (Part 1) and on characteristics of MetS in the dams and their second litter (L2) offspring (Part 2) were examined. Part 1 (studies 1 and 2) hypothesis was that the maternal HV-diet alters tissue fatty acid (FA) concentrations, expression of peroxisome-proliferator-activated receptors (PPARs) genes, and their regulation of metabolism in the offspring, favoring insulin resistance. Part 2 (studies 3 and 4) hypothesis was that high-vitamin intakes during the first pregnancy increase weight gain, food intake and markers of MetS in both the dams and their litters. In all experiments, dams were fed the RV or HV-diet. In study 4, a high-folic-acid-diet (HFol, RV+10-fold folic acid) was added. In studies 1 and 2, the offspring were weaned to an obesogenic diet. The HV-diet affected tissue FA concentrations (study 1), increased muscle PPAR-ɑ mRNA levels and uncoupled relationships between hepatic PPAR-γ mRNA levels and insulin resistance (study 2) in male offspring. In study 3, dams fed the HV-diet during the first pregnancy were maintained on the RV-diet and then mated again after 12wk. Their litters were fed the RV-diet. The HV-diet increased weight gain and food intake of both dams and L2, and insulin resistance in their offspring. In study 4, both HV and HFol-diets increased post-weaning weight gain, but differed in their effects on biomarkers of food intake regulation. In conclusion, feeding the HV-diet during the first pregnancy increases post-weaning body weight and food intake in Wistar rat dams, uncouples tissue regulation of glucose metabolism and promotes characteristics of MetS in their litters. Folic acid is not the only vitamin involved.

Salt iodization is widely available throughout the developing world. Incorporating other micronutrients into the existing salt iodization process could prevent multiple deficiencies. The thesis objective was to develop a stable formulation of salt dual fortified with folic acid and iodine, using a single solution that could be sprayed on. The micronutrients needed to be fully solubilized and stable in solution for at least one month. In the absence of an alkaline environment or antioxidant, iodine losses occur most likely due to the oxidation of folic acid by potassium iodate. Optimal salt formulations were prepared by spraying a pH 9 carbonate-bicarbonate buffer solution containing folic acid and iodine dissolved at 0.35% (w/v) each. Acceptable micronutrient retentions of > 90% were observed in refined salt after 6 months of storage at 45°C/60% relative humidity. Further investigations into increasing the concentration of iodine and folic acid in the spray solution are recommended.

High vitamin (HV), AIN-93G diet with ten-fold the regular amount of vitamins (RV), consumed by pregnant Wistar rats increases characteristics of metabolic syndrome (MetS) in their first litters. Therefore, the effects of the maternal HV-diet on tissue mechanisms regulating insulin resistance in offspring (Part 1) and on characteristics of MetS in the dams and their second litter (L2) offspring (Part 2) were examined. Part 1 (studies 1 and 2) hypothesis was that the maternal HV-diet alters tissue fatty acid (FA) concentrations, expression of peroxisome-proliferator-activated receptors (PPARs) genes, and their regulation of metabolism in the offspring, favoring insulin resistance. Part 2 (studies 3 and 4) hypothesis was that high-vitamin intakes during the first pregnancy increase weight gain, food intake and markers of MetS in both the dams and their litters. In all experiments, dams were fed the RV or HV-diet. In study 4, a high-folic-acid-diet (HFol, RV+10-fold folic acid) was added. In studies 1 and 2, the offspring were weaned to an obesogenic diet. The HV-diet affected tissue FA concentrations (study 1), increased muscle PPAR-ɑ mRNA levels and uncoupled relationships between hepatic PPAR-γ mRNA levels and insulin resistance (study 2) in male offspring. In study 3, dams fed the HV-diet during the first pregnancy were maintained on the RV-diet and then mated again after 12wk. Their litters were fed the RV-diet. The HV-diet increased weight gain and food intake of both dams and L2, and insulin resistance in their offspring. In study 4, both HV and HFol-diets increased post-weaning weight gain, but differed in their effects on biomarkers of food intake regulation. In conclusion, feeding the HV-diet during the first pregnancy increases post-weaning body weight and food intake in Wistar rat dams, uncouples tissue regulation of glucose metabolism and promotes characteristics of MetS in their litters. Folic acid is not the only vitamin involved.

Salt iodization is widely available throughout the developing world. Incorporating other micronutrients into the existing salt iodization process could prevent multiple deficiencies. The thesis objective was to develop a stable formulation of salt dual fortified with folic acid and iodine, using a single solution that could be sprayed on. The micronutrients needed to be fully solubilized and stable in solution for at least one month. In the absence of an alkaline environment or antioxidant, iodine losses occur most likely due to the oxidation of folic acid by potassium iodate. Optimal salt formulations were prepared by spraying a pH 9 carbonate-bicarbonate buffer solution containing folic acid and iodine dissolved at 0.35% (w/v) each. Acceptable micronutrient retentions of > 90% were observed in refined salt after 6 months of storage at 45°C/60% relative humidity. Further investigations into increasing the concentration of iodine and folic acid in the spray solution are recommended.

No description available.

Errata pasted onto t.p. verso. / Bibliography: leaves 129-148. / 148 leaves : [3] col. ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Investigates the relationship between folate states and colorectal cancer risk using the rat AOM intestinal cancer model. Results indicate that folate status can play an important role in modulating colorectal carcinogenesis. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 2000

Thesis (M.Sc. (Tropical Medicine))--Mahidol University, 1978.