No description available.

No description available.

No description available.

No description available.

Fibroblasts from patients with functional methionine synthase deficiency can be divided into 2 complementation classes, cblE and cblG. Both have low levels of intracellular methylcobalamin. Both groups also demonstrate low levels of incorporation of label from 5-methyltetrahydrofolate into macromolecules. Under standard reducing conditions, methionine synthase specific activity is normal in cblE fibroblast extracts, but is low in cblG fibroblast extracts. Seven cblE and seven out of ten cblG cell lines demonstrate levels of accumulation of ($ sp{57}$Co) CN-Cbl in fibroblasts comparable to that of control cells. They exhibit similar proportions of label associated with the two intracellular cobalamin binders, methylmalonyl-CoA mutase and methionine synthase. The remaining three cblG cell lines exhibit a lower level of cobalamin accumulation, and demonstrate a lack of cobalamin association with the enzyme methionine synthase. The specific activity of methionine synthase is almost undetectable in the three cblG cell lines that showed no such association. These results demonstrate heterogeneity within the cblG group and suggest that the defect in cblG affects the methionine synthase apoenzyme.

No description available.

Previously, ADH1Cc.-64T>C was shown to have an association with intramuscular fat (IMF) in the longissimus thoracis (LT) muscle when vitamin A was limited in finishing rations of beef steers. The purpose of the current study was to determine the optimum vitamin A supplementation level, in combination with ADH1C genotype, to increase IMF of the LT muscle. Forty-five TT, 45 CT and 27 CC cross-bred steers, black in colour, were backgrounded on a commercial ration containing 3360 IU vitamin A/kg DM. During finishing the steers were randomly assigned to one of three vitamin A treatments at 25, 50 and 75% of the NRC recommendation of 2200 IU/kg DM. Treatments were administered via an oral bolus. Carcass quality was evaluated and a sample from the LT muscle was collected for analysis of IMF. A treatment x genotype interaction (P=0.04) was observed for IMF; TT steers on the 75% treatment had higher IMF relative to CT and CC steers on the same treatment. Intramuscular fat was also higher for TT steers on the 75% treatment in comparison to TT steers on the 25% treatment. Eighty-four percent of the steers graded Canada AAA. Western blot analysis showed that TT steers had higher (P=0.02) ADH1C levels in hepatic tissue. Previously, TT steers had increased IMF when fed limited vitamin A. In the current study the lack of variation between treatments and genotypes at the lower vitamin A treatment levels was likely due to the majority of the steers grading Canada AAA (USDA Choice). However, the western blot data supports that TT steers are expected to have higher IMF deposition, due to an increase production of ADH1C.

Epidemiologic, laboratory and clinical data suggest that vitamin D plays a favourable role in the prevention and prognosis of prostate cancer (PCa) and other malignancies. However, the metabolism and function of vitamin D in tissues beyond those involved in bone metabolism are poorly understood. The objective of this thesis was to investigate whether higher levels of vitamin D consumed orally or achieved in the circulation result in increased concentrations of vitamin D metabolites in human tissue, and how this affects cellular biology. The hallmark of this work is a randomized clinical trial of oral vitamin D3 (400-, 10,000-, or 40,000 IU/d) in PCa patients to evaluate the effects of supplementation on prostatic vitamin D metabolism and on PCa pathology. Various methods to measure vitamin D metabolites in serum were evaluated and modified to allow for measurement of these metabolites in tissue. Ultimately, I developed a robust tissue extraction method coupled to enzyme immunoassay and liquid chromatography-tandem mass spectrometry for measurement of calcitriol hormone, as well as 25-hydroxyvitamin D (25(OH)D) and 24,25-dihydroxyvitamin D (24,25(OH)2D), respectively. Human colon tissue was analyzed first and found to contain calcitriol at physiologically relevant concentrations partly determined by serum calcitriol, with some evidence of local colonic synthesis. In the clinical trial, prostate tissue and serum levels of calcitriol, 25(OH)D and 24,25(OH)2D increased dose-dependently (p<0.02) without adverse side effects. The level of calcitriol attained in prostate tissue was inversely associated with the expression of Ki67 protein, a proliferation marker (p<0.05). Serum parathyroid hormone (PTH) and prostate specific antigen (PSA) declined from baseline in the combined higher-dose groups (10,000-40,000 IU/d) (p<0.02). We provide clinical trial evidence that prostatic vitamin D metabolism can be modulated in vivo by oral consumption of vitamin D3. Higher prostate calcitriol and vitamin D doses also showed suggestion of clinical benefit, including lowered Ki67 expression and modest reductions in serum PSA and PTH. Further studies are needed to validate the potential utility of dietary vitamin D3 supplementation in cancer prevention and therapy.

Epidemiologic, laboratory and clinical data suggest that vitamin D plays a favourable role in the prevention and prognosis of prostate cancer (PCa) and other malignancies. However, the metabolism and function of vitamin D in tissues beyond those involved in bone metabolism are poorly understood. The objective of this thesis was to investigate whether higher levels of vitamin D consumed orally or achieved in the circulation result in increased concentrations of vitamin D metabolites in human tissue, and how this affects cellular biology. The hallmark of this work is a randomized clinical trial of oral vitamin D3 (400-, 10,000-, or 40,000 IU/d) in PCa patients to evaluate the effects of supplementation on prostatic vitamin D metabolism and on PCa pathology. Various methods to measure vitamin D metabolites in serum were evaluated and modified to allow for measurement of these metabolites in tissue. Ultimately, I developed a robust tissue extraction method coupled to enzyme immunoassay and liquid chromatography-tandem mass spectrometry for measurement of calcitriol hormone, as well as 25-hydroxyvitamin D (25(OH)D) and 24,25-dihydroxyvitamin D (24,25(OH)2D), respectively. Human colon tissue was analyzed first and found to contain calcitriol at physiologically relevant concentrations partly determined by serum calcitriol, with some evidence of local colonic synthesis. In the clinical trial, prostate tissue and serum levels of calcitriol, 25(OH)D and 24,25(OH)2D increased dose-dependently (p<0.02) without adverse side effects. The level of calcitriol attained in prostate tissue was inversely associated with the expression of Ki67 protein, a proliferation marker (p<0.05). Serum parathyroid hormone (PTH) and prostate specific antigen (PSA) declined from baseline in the combined higher-dose groups (10,000-40,000 IU/d) (p<0.02). We provide clinical trial evidence that prostatic vitamin D metabolism can be modulated in vivo by oral consumption of vitamin D3. Higher prostate calcitriol and vitamin D doses also showed suggestion of clinical benefit, including lowered Ki67 expression and modest reductions in serum PSA and PTH. Further studies are needed to validate the potential utility of dietary vitamin D3 supplementation in cancer prevention and therapy.

The synthesis of several fluorinated analogues of thiamine and precursors has been undertaken, all of which exhibit weak activity against E.Coli. A route to 3-fluoro-(R)-alanine has been explored. The first synthesis of the enantiomeric fluorosuccinic acids has been accomplished, and a stereochemical analysis has shown that synthesis via esterified malate precursors using diethylamino- sulphurtrifluoride gives inversion of configuration, whereas prep- aration via aspartic acid and polyhydrogen fluoride in pyridine gives retention of configuration. The circular dichroism spectra of the fluorosuccinic acids are anomalous, being the first example of such behaviour in a-substituted carboxylic acids and derivatives. A previously assigned configuration of a Pseudomonal metabolite ( + )- fluorosuccinic acid has been corrected. The fluorosuccinic acids are not substrates for malic enzyme, but are potent inhibitors of fumarase. Using (2S)-fluorosuccinic acid as a mechanistic probe the fumarase reaction is identified as a bimolecular E2 elimination. A stereochemical analysis of enzymic phosphoryl transfer reactions using CD spectroscopy has been investigated, and a new synthesis of inorganic pyrophosphate used to prepare P₁=[(S)- ¹⁶0, ¹⁷0, ¹⁸0]-pyrophosphate whose ³¹P n.m.r. spectrum is discussed. The first example of 31 p- 17 o coupling as observed by 31 P n.m.r. is demonstrated.- The magnitude of the ³¹P- ¹⁸0 isotope shift is dependent on the nature of the phosphorus to oxygen bond. An elegant method for establishing the isotopic configuration of oxygen chiral phosphates by n.m.r. has been developed, requiring the synthesis of D-glucose-6[(S) - ¹⁶0, ¹⁷0, ¹⁸0]-phosphate, adenosine-5'- [(S)- ¹⁶0, ¹⁷0, ¹⁸0]-phosphate, and their six membered cyclic phosphate triesters. Chemical cyclisation of these molecules occurs both with racemisation and inversion of configuration at phosphorus. The chemical synthesis of adenosine-5'[ (S) - ¹⁶0, ¹⁷0, ¹⁸0]- triphosphate has been achieved and exploited to demonstrate that hexokinase phosphoryl transfer proceeds with inversion of config- uration at phosphorus. Pyruvate kinase phosphoryl transfer from 2 [(S)- ¹⁶0, ¹⁷0, ¹⁸0]-phospho- (R)-glycerate also proceeds with inversion of configuration, as does phosphofructokinase phosphoryl transfer from sn-glycerol-3[ (5)- ¹⁶0, ¹⁷0, ¹⁸0]-phosphate.