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Previous studies have found that smoking may have a negative effect on vitamin B-6 indices and have demonstrated a possible association between smoking and depressed plasma pyridoxal-5'-phosphate (PLP) concentration. Individuals with plasma PLP values below the adequate level of 30 nmoles/L might benefit from consumption of vitamin B-6 supplements, but no data are available on vitamin B-6 status in smokers consuming a controlled vitamin B-6 intake and receiving a vitamin B-6 supplement. The objectives of this research were to assess vitamin B-6 status in smokers as compared to non-smokers receiving a controlled diet and to evaluate the effect of an oral vitamin B-6 supplementation in these subjects. The vitamin B-6 (B-6) status of 5 (four males / one female) smokers (S) and 4 (three males / one female) non-smokers (NS) was assessed. A constant diet was fed for 20 days and provided 1.95 mg of B-6 or 1.65 mg of B-6 for males and females, respectively. For the last 10 days, an additional 2-mg of pyridoxine (PN) was given daily. Blood samples were collected on days 1.7, 11.14 and 21; and 24 hour urine samples were collected daily. Urinary 4-pyridoxic acid (4-PA) and total B-6 (UB6) excretion, plasma B-6 vitamers (PLP, PN, pyridoxal and 4-PA) and red blood cell PLP (RBC PLP) concentrations, as well as plasma alkaline phosphatase activity (APA) were determined. Mean plasma PLP, 4-PA, and RBC PLP concentrations were significantly lower (P [less than or equal to] 0.05) at all time points in S compared to NS. With a daily supplement of 2-mg vitamin B-6, the mean plasma PLP concentration of S increased 85.8% but was 48.5% lower than that of NS consuming 1.65-1.95 mg/d of B-6. Mean plasma pyridoxal concentrations were not different between S and NS before and after supplementation. Excretion of 4-PA was not significantly different between S and NS, but the mean values of 4-PA excretion were consistently greater in NS compared to that of S throughout the 20-day study. The percent of ingested B-6 excreted as 4-PA for the S and NS was 38 and 49 in the non-supplemented period, and 47 and 53 in the supplemented period, respectively, indicating that non-smokers excreted more 4-PA than smokers. However, the difference in 4-PA excretion between S and NS was not significantly different both before and after supplementation (P>0.05). In addition, there was no significant difference between S and NS for plasma PN concentration, AP, and UB6 excretion for both periods. Results suggested an adverse effect of smoking on B-6 metabolism, thus an increased requirement of vitamin B-6 in smokers. A 2-mg PN supplement was sufficient to bring the concentration of plasma PLP in smokers to the level suggested as adequate, but it didn't bring it to the level of non-smokers. / Graduation date: 1999

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by Priscilla Miu-kuen Poon. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1991. / Includes bibliographical references. / ACKNOWLEDGEMENT --- p.1 / ABSTRACT --- p.2 / CONTENTS / Chapter 1. --- INTRODUCTION --- p.4 / Chapter 1.1 --- Discovery of vitamin D / Chapter 1.2 --- Bioavailability of vitamin D and its metabolites / Chapter 1.3 --- Metabolism of vitamin D and its metabolites / Chapter 1.4 --- Mode of action of vitamin D / Chapter 1.5 --- Vitamin D-related diseases / Chapter 2. --- METHODS OF MEASURING VITAMIN D AND ITS METABOLITES --- p.32 / Chapter 2.1 --- Deproteinization / Chapter 2.2 --- Extraction / Chapter 2.3 --- Separation / Chapter 2.4 --- Quantitation / Chapter 3. --- OBJECTIVES --- p.51 / Chapter 4. --- MATERIALS & METHODS --- p.52 / Chapter 4.1 --- Materials / Chapter 4.2 --- General methods / Chapter 4.3 --- Blood collection / Chapter 4.4 --- Radioreceptor assay / Chapter 4.5 --- Serum treatment / Chapter 4.6 --- High Performance Liquid Chromatography (HPLC) / Chapter 4.7 --- Gas Chromatography-Mass Spectrometry (GC-MS) / Chapter 4.8 --- "Serum 1α,25-dihydroxyvitamin D3 analysis" / Chapter 4.9 --- Application of the established GC-MS method / Chapter 4.10 --- Study on hypercalcaemia of tuberculosis / Chapter 5. --- RESULTS --- p.66 / Chapter 5.1 --- Analysis of vitamin D3 standard / Chapter 5.2 --- "Analysis of 1α,25-dihydroxyvitamin D3 standard" / Chapter 5.3 --- Separation of vitamin D3 metabolites / Chapter 5.4 --- "Analysis of lα,25-dihydroxyvitamin D3 in serum samples" / Chapter 5.5 --- Study on hypercalcaemia of tuberculosis / Chapter 6. --- DISCUSSIONS --- p.118 / Chapter 6.1 --- Derivatization / Chapter 6.2 --- Optimization of GC-MS parameters / Chapter 6.3 --- Sample pre-treatment / Chapter 6.4 --- "GC-MS analysis of serum lα,25-dihydroxyvitamin D3" / Chapter 6.5 --- Study on hypercalcaemia of tuberculosis / Chapter 7. --- CONCLUSION --- p.129 / LIST OF ABBREVIATIONS --- p.131 / LIST OF FIGURES --- p.134 / LIST OF TABLES --- p.137 / REFERENCES --- p.139

Several of the inborn errors of vitamin B$ sb{12}$ (cobalamin, Cbl) metabolism (cblC, cblD, cblE, cblF, cblG) are associated with homocystinuria and hypomethioninemia due to a functional deficiency of the cytoplasmic enzyme methionine synthase which requires methylcobalamin (MeCbl) as a cofactor. We compared the growth of cultured fibroblasts from controls with those from patients with a selective deficiency of MeCbl (cblE and cblG) and with those from patients with a defect in both MeCbl and adenosylcobalamin (AdoCbl) (cblC, cblD and cblF). Cells were grown in methionine and folic acid free media and in fully supplemented medium. Control cells were able to grow in the deficient medium supplied with homocysteine, cobalamin and folate, while mutant cells were not, due to their inability to synthesize methionine from its immediate metabolic precursor, homocysteine. This differential growth is useful for screening for genetic defects of methionine biosynthesis. Moreover, by correcting methionine auxotrophy in these cells, it may be possible to isolate genes which code for the products that are deficient in these disorders.

Methionine synthase (MS) is a vitamin B12(cobalamin;cbl) dependent enzyme that catalyses the methylation of homocysteine to methionine. It uses methyl-cbl as coenzyme and in ethyl tetrahydrofolate as the methyl donor. Methionine sythase reductase (MSR) maintains MS in it active state using S-adenosyl methionine as the methyl donor. Functional MS deficiency may occur as a result of a defect in either enzyme. Patients with this disorder have been classified into two complemetation groups according to which protein is defective: cblG patients are deficient in MS and cblE patients in MSR. A subset of cblG, known as cblG variant, is unique in showing barely detectable MS activity and failure of cbl incorporation into MS in patient fibroblasts. I report the mutations responsible for three cblG variant patients, two of them siblings, and connect their phenotype to lack of protein expression. I also report the cloning of the MSR cDNA, aided by confirming the identity of the cDNA through the discovery of two deleterious mutations in three cblE patients. These findings contribute to the overall understanding of functional MS deficiency.

The first three studies in this thesis address the mechanism for the aberrant fall in serum 1,25-dihydroxyvitamin D$ sb3$ (1,25-(OH)$ sb2$D$ sb3 rbrack$ and increase in renal 1,25-(OH)$ sb2$D$ sb3$-24-hydroxylase(24-hydroxylase) activity in X-linked hypophosphatemic mice (Hyp). The 24-hydroxylase is the first enzyme in the C-24 oxidation pathway that degrades the vitamin D hormone to its final inactivation product, calcitroic acid. We demonstrated that: (i) the aberrant increase in 24-hydroxylase activity in Pi-deprived Hyp mice is specific to the kidney and is the result of an increase in enzyme Vmax, immunoreactive protein and mRNA abundance; (ii) the increase in 24-hydroxylase mRNA in both Pi-deprived Hyp mice and 1,25-(0H)$ sb2$D$ sb3$-treated normal littermates can be ascribed to an increase in the transcriptional activity of the 24-hydroxylase gene; (iii) 24-hydroxylase transcripts in normal mice, Pi-deprived Hyp and normal mice and 1,25-(OH)$ sb2$D$ sb3$-treated normal mice are localized to the proximal tubule by in situ hybridization; and (iv) recombinant human growth hormone administration normalizes the aberrant increase in 24-hydroxylase but that this response is not sufficient to correct serum 1,25-(OH)$ sb2$D$ sb3$ levels in Pi-deprived Hyp mice. / The fourth study addresses the mechanism whereby EB 1089, an analogue of 1,25-(OH)$ sb2$D$ sb3,$ is less calcemic than the vitamin D hormone, while being more potent in its antiproliferative action. We demonstrate that: (i) EB 1089 has a 50-fold lower affinity than 1,25-(OH)$ sb2$D$ sb3$ for the vitamin D catabolic enzyme, 24-hydroxylase; and (ii) EB 1089 and 1,25-(OH)$ sb2$D$ sb3$ exhibit tissue-specific differences in vitamin D receptor-mediated responses in vivo that may be ascribed, at least in part, to differences in binding affinities for the vitamin D receptor.

Doctorat en sciences médicales / info:eu-repo/semantics/nonPublished