The plasma vitamin D binding protein (DBP) is an a-glycoprotein, synthesised and secreted by the liver, which binds specifically vitamin D and its metabolites. The DBP molecule, has a single high affinity binding site for its ligands, and is present in blood in concentrations about 1000-fold greater than the sum of all its vitamin D ligands. Previous studies have not found any change in the concentration of DBP related to various derangements in mineral homeostasis. Therefore the general view is that DBP has a passive role in the physiology of vitamin D and its metabolites, and simply acts to solubilise and transport these hydrophobic ligands in the aqueous extracellular fluid. However, differences which have been described in its affinity for various vitamin D metabolites suggest that there have been evolutionary influences on the properties of this protein. Furthermore, plasma DBP concentration has been found to change in response to a number of physiological factors, such as changing sex steroid hormone secretion. The aim of the studies presented in this thesis was to investigate variation in the plasma concentration of the DBP in a range of vertebrate species, and in response to a variety of physiological factors. The results suggest that DBP may have an active role in regulating the bioavailability, and hence the utilisation and metabolism of its ligands. DBP concentration has traditionally been measured using immunological techniques. These techniques, although fast and simple, have a number of draw-backs which can be overcome by the use of assays which rely upon functional aspects of the DBP. A saturation binding assay was modified from those described previously. Using this technique, it was found that both the circulating concentration of the DBP and its affinity for 25-hydroxyvitamin D3 (25(OH)D3) varied significantly among a wide range of species of reptiles and birds. This variation did not reflect phylogenetic relationships among the study species, suggesting that the variation was more likely to be the result of selective pressure in response to individual ecological or physiological circumstance, rather than to random mutation. In support of this, both the plasma concentration of DBP, and its affinity for 25(OH)D3 were significantly associated with a number of ecological factors which might be considered to have some significance to vitamin D and calcium homeostasis. In addition, comparative binding data suggests that the ability of the DBP to bind 25-hydroxyvitamin D2 with equal affinity to 25(OH)D3 is an evolutionary innovation of mammalian vertebrates. In order to extend the idea of genetic variation in the concentration and affinity of plasma DBP, two strains of broiler (meat-type) chickens were studied. It was found that both the concentration and the affinity of plasma DBP for 25(OH)D3 was characteristic for each strain, emphasising the sensitivity of DBP to genetic variation. A number of factors have been found to modulate the genetically determined plasma concentration of DBP. Deficiencies of dietary protein and dietary energy, and variation in concentrations of sex steroids were found to affect the circulating concentration of DBP. However, species differences were still apparent, suggesting that the sensitivity of DBP to these physiological modifiers may have developed independently in different species, and may be secondary to genetic determinants of DBP properties. The plasma DBP concentration and specific binding affinity both determine the availability of its ligands for cellular uptake. It is likely that this process is complex, and involves a combination of protein mediated and non-mediated uptake events. This makes DBP a potentially important determinant of the biological actions of its ligands. The studies in this thesis have produced two main lines of argument supporting an active role for DBP in the regulation of vitamin D metabolism and utilisation. The first is that genetic variation in the properties of plasma DBP appears to be genetically determined, and is selected for, both at the between-species, and the within-species level, than it is to random mutation. Secondly, the ability of physiological and environmental factors to modify the circulating concentration of DBP suggests that this protein is responsive to homeostatic processes. It is proposed that DBP is an active regulator of the physiological economy of vitamin D and its metabolites by being itself regulated by a number of genetic and non-genetic factors.

Phytol aus dem Chlorophyllabbau ist limitierend für die Tocopherol (Vitamin E)-Synthese Als Bestandteil von Chlorophyll ist Phytol das am häufigsten vorkommende Isoprenoid in der Biosphäre. Große Mengen an Chlorophyll werden jährlich degradiert und dabei wird Phytol freigesetzt, über dessen Verbleib jedoch wenig bekannt ist. Es sollte der Nachweis erbracht werden, dass im Zuge des Chlorophyllabbaus hydrolysiertes Phytol Eingang in die Synthese anderer Phytylderivate findet. Während der Gehalt an Tocopherol, Chlorophyll und Fettsäurephytylester entwicklungs- bzw. seneszenzabhängig ist, bleibt der Gehalt an Phyllochinon etwa gleich. Auch in Samen ist der Gehalt von Tocopherol, Chlorophyll und Fettsäurephytylester entwicklungsabhängig. Es wurde gefolgert, dass nur die Synthesen von Tocopherol und Fettsäurephytylester während des Chlorophyllabbaus stimuliert werden. Daher sollten Mutanten analysiert werden, welche im Chlorophyllabbau inhibiert sind. Da Chlorophyllase den ersten Schritt des Chlorophyllabbaus katalysiert, wurden zwei unabhängige T-DNA-Insertionsmutanten für Chlorophyllase1 (CHL1) und eine T-DNA-Insertionsmutante für Chlorophyllase2 (CHL2) identifiziert und eine chl1-1chl2-Doppelmutante erzeugt. Die Analyse der Chlorophyllidanteile ergab eine im Vergleich zum Wildtyp starke Reduktion in den chl1-Mutanten, während der Chlorophyllidanteil von chl2 ähnlich hoch dem Wildtyp ist. Der Chlorophyllidanteil sich entwickelnder chl1-1chl2-Pflanzen nahm in der Seneszenz zu. Die Chlorophyllasemutanten zeigten kein verändertes Seneszenzverhalten im Vergleich zu den Wildtypen. Ferner konnte in den chl1-Linien nur geringfügig weniger Tocopherol und Fettsäurephytylester als in den Wildtypen nachgewiesen werden. Auch der Tocopherolgehalt der Samen war in den Chlorophyllasemutanten unverändert zu den Wildtypen. Aufgrund dessen wurde gefolgert, dass neben den Chorophyllasen CHL1 und CHL2 weitere Chlorophyllhydrolasen in Samen und Blättern von Arabidopsis existieren. Daher wurde auf andere Mutanten zurückgegriffen, in denen der Chlorophyllabbau stark inhibiert ist und die Seneszenz nach Dunkelinkubation im Vergleich zum Wildtyp deutlich verzögert ist. Eine deutliche Korrelation zwischen vermindertem Chlorophyllabbau und Gehalt an Tocopherol und Fettsäurephytylester konnte in den staygreen-Mutanten pao1 und zwei unabhängigen SGR (staygreen)-RNAi-Linien nachgewiesen werden. Damit konnte eindeutig gezeigt werden, dass die Synthese von Tocopherol und der Fettsäurephytylester durch die Chlorophyllhydrolyse induziert wird. Es wurde gefolgert, dass vor allem unter Seneszenz- bzw. Stressbedingungen dieser alternative Syntheseweg von Phytol eine Rolle spielt. Dennoch kommt der Phytylsynthese durch die de novo-Isoprenoidsynthese auch eine Bedeutung zu. Nach Behandlung von stickstoffmangelgestressten Wildtyppflanzen mit dem Inhibitor Fosmidomycin, welcher die plastidäre de novo-Isoprenoidsynthese hemmt, war der Tocopherolgehalt gegenüber stickstoffmangelgestressten Kontrollpflanzen stark reduziert. Ferner konnte eine T-DNA-Insertionsmutante der Geranylgeranylreduktase (GGR) identifiziert werden. Diese Mutante kann nur auf Nährmedium überleben, hat nur wenige grüne Blätter und bildet keine Samen. Es konnte kein Phyllochinon, Chlorophyll und keine Fettsäurephytylester, jedoch geringe Mengen Tocopherol nachgewiesen werden. Der Resttocopherolgehalt wird auf die Nebenaktivität einer anderen Reduktase zurückgeführt. Weiterhin wurde nur das Geranylgeranylderivat des Chlorophylls identifiziert. Diese Ergebnisse erlauben den Schluss, dass die phytylgruppenübertragenen Enzyme der Tocopherol-, Phyllochinon- und Fettsäurephytylestersynthese eine hohe Substratspezifität für die Phytylgruppe aufweisen. Nach Fütterung von Phytol konnte in ggr Tocopherol und Chlorophyll bestimmt werden. Aufgrund dessen kann gefolgert werden, dass Chlorophyllsynthetase aus Arabidopsis sowohl Geranylgeranyl-, als auch Phytylpyrophosphat als Substrat nutzen kann und damit ein breiteres Substratspektrum aufweist. / Phytol from chlorophyll degradation is limiting for tocopherol (vitamin E)-synthesis As a part of the chlorophyll molecule phytol belongs to the most abundant isoprenoid of the biosphere. Huge amounts of chlorophyll are degraded annually. During this process phytol is released, but only little is known about the fate of phytol. The goal of the project was to provide evidence that during chlorophyll degradation released phytol enters the pathway of the synthesis of further phytyl derivatives. While the content of tocopherol, chlorophyll and fatty acid phytyl esters are growth and stress related the content of phylloquinone does not change during development or under stress conditions. Also in seeds the content of these phytyl derivates are dependent on development. Hence only tocopherol and fatty acid phytyl ester synthesis are induced during chlorophyll degradation. Therefore mutants were analysed that are inhibited in chlorophyll degradation. Chorophyllase catalyses the first step during chlorophyll degradation. Two independent T-DNA insertion mutants of Chlorophyllase1 (CHL1) and one for Chlorophyllase2 (CHL2) were identified. Furthermore chl1-1 and chl2 were crossed to produce the chl1-1chl2 double mutant. The mutation resulted in a strong reduction of the chlorophyllide fraction in chl1 mutants while the chlorophyllide fraction of chl2 was similar to wild type. The chlorophyllide fraction in developing chl1-1chl2 plants increased during senescence. For all chlorophyllase mutants no retardation of senescence was observed. Compared to wild type only marginal reductions in tocopherol and fatty acid phytyl ester contents could be observed for the chl1 mutants. The seed tocopherol content of the chlorophyllase mutants was similar to wild type. Therefore, it was concluded that in leaves and seeds of Arabidopsis besides CHL1 and CHL2 further chlorophyll hydrolases exist that induce chlorophyll degradation. Thus, staygreen mutants exhibiting strongly inhibited chlorophyll degradation were analysed. Compared to wild type the staygreen mutants pao1 and two independent SGR (staygreen)-RNAi-lines show a strong retardation of senescence under dark incubation. A clear correlation between reduced chlorophyll degradation and tocopherol and fatty acid phytyl ester content could be demonstrated. With this it was possible to verify that tocopherol and fatty acid phytyl ester synthesis are induced by chlorophyll hydrolysis. This alternative pathway seems to play an important role in particular under stress and senescence conditions. Nevertheless, after application of Fosmidomycin, an inhibitor of the plastidic de novo isoprenoid synthesis pathway, to nitrogen starved wild type plants the tocopherol content was strongly reduced compared to nitrogen starved control plants. Therefore, also the plastidic de novo isoprenoid synthesis plays a significant role for tocopherol synthesis. Moreover, a T-DNA insertion mutant for Geranylgeranyl reductase (ggr) was identified and isolated. This mutant can survive only on nutrition medium, contains only a few green leaves and produces no seeds. There was no phylloquinone, chlorophyll and fatty acid phytyl ester detectable, but minor amounts of tocopherol. The residual amounts of tocopherol were attributed to side activities of another reductase. Obviously, the phytyl transferring enzymes of tocopherol, phylloquinone and fatty acid phytyl ester synthesis exhibit a strong substrate specificity of the phytyl group. After feeding phytol to ggr tocopherol and chlorophyll were detectable in this mutant. Therefore, it was concluded that chlorophyll synthetase from Arabidopsis can use geranylgeranyl pyrophosphate as well as phytyl pyrophosphate as substrates.

Thesis (M.S.)--University of Wyoming, 2008. / Title from PDF title page (viewed on Dec. 4, 2009). Includes bibliographical references (p. 75-88).

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 163-189).

Thesis (M. Phil.)--University of Hong Kong, 1987.

Despite the medical advances in recent decades, cardiovascular disease (CVD) remains one of the leading causes of mortality in most developing countries. Ongoing efforts have been focused on evaluating new strategies targeting on novel risk factors. Vitamin D deficiency, a previously neglected condition, has recently attracted much attention from the scientific community with its potential extra-skeletal effects. There is accumulating evidence from epidemiological studies that a suboptimal 25-hydroxyvitamin D [25(OH)D] level is associated with all-cause and cardiovascular mortality, increased risk of coronary heart disease, stroke and peripheral vascular disease, and various traditional CVD risk factors including hypertension, diabetes mellitus (DM) and metabolic syndrome. Several theories have been proposed to explain these relationships but none receive universal recognition. There is recent laboratory evidence that vitamin D may exert specific effects in patients with DM. However, relationships between vitamin D deficiency and supplementation on vascular function in this group of patients are unclear. In this dissertation, I sought to explore the effects of vitamin D deficiency on vascular function in patients with type II DM in a cross-sectional study. In the later part, the results of a randomized controlled trial investigating the effects of daily vitamin D supplementation in type II DM patients are presented and discussed. The cross-sectional study (Chapter 3) investigated the association of vitamin D status with endothelial function as measured by brachial flow-mediated dilation (FMD) and circulating endothelial progenitor cell (EPC) numbers in 280 patients with type II DM. The results showed that suboptimal vitamin D status was more common among patients with DM. Furthermore, patients with vitamin D deficiency had significantly lower brachial FMD (mean difference = -1.43%, 95% CI: -2.31 to -0.55, P = 0.001) and CD133/KDR+ EPC counts (mean difference = -0.12%, 95% CI: -0.21 to -0.02, P = 0.022) than those with sufficient vitamin D after adjustment for age, sex and cardiovascular risk factors, including HbA1c levels. Based on these positive results, the objectives of the randomized controlled trial (Chapter 4) were to study and confirm the effects of daily oral vitamin D supplementation on the vascular function in this group of patients. Over a 12-week period, 100 DM patients with suboptimal vitamin D status were randomized to receive 5,000 IU/day vitamin D or placebo. There were no reported adverse events including hypercalcemia, although a slight increase in serum ionized calcium (treatment effect 0.037 mmol/L, P = 0.018) was recorded in the vitamin D group. Despite a significant improvement in serum 25(OH)D in the treatment group, supplementation of vitamin D did not result in any significant improvement in vascular function as determined by FMD, circulating EPC count or arterial stiffness (all P > 0.05). Furthermore, the serum level of high-sensitivity C-reactive protein, oxidative stress markers, low- and high-density lipoprotein and glycated haemoglobin were also similar between two groups (all P > 0.05). The results of this study did not support a therapeutic role of supplementation with vitamin D for cardiovascular benefits. In conclusion, the results of these studies demonstrated that deficiency of vitamin D was associated with worse vascular function in patients with type II DM. However, vitamin D supplementation did not result in any significant benefits on vascular function or improvement in traditional CVD risk factors in DM patients. Further large clinical trials on vitamin D supplementation in patients with DM using clinical outcomes rather than surrogate CVD markers are necessary to confirm its benefits. / published_or_final_version / Medicine / Master / Master of Research in Medicine

published_or_final_version / Biochemistry / Master / Master of Philosophy

No description available.

Vitamin A has well recognised benefits for the reduction in severity of diarrhoeal episodes but the impact of therapeutic doses given during diarrhoea on the biochemical and clinical outcomes is less clear. In this study these potential therapeutic benefits were investigated to establish the optimum time for vitamin A supplementation to improve vitamin A status. Establishing the optimum time for vitamin A supplementation during an infectious stage would improve cost effectiveness and clinical benefit. Young children (174) between the ages of 3 and 60 months with severe diarrhoea were randomised in a double - blinded placebo controlled trial into one of 2 groups. The 1 st group received 60 mg of retinol as retinyl palmitate on admission during the acute diarrhoeal stage. The 2nd group received the same dose of vitamin A once symptoms had resolved, usually between 3 - 7 days. At each of these two time points, children not receiving vitamin A were given an identical placebo dose. Baseline (day 0) and day 3 serum samples were collected for vitamin A, retinol binding protein (RBP) and other biochemical markers. At four and eight weeks after discharge both morbidity and weight gain were recorded. The modified dose response test (MRDR) was conducted at the eight - week follow - up to estimate vitamin A liver stores. Initially, most of the children presented with watery diarrhoea and dehydration and were clinically very ill. At day 3 plasma retinol concentrations improved in both groups viz. from 0.57umol/L to 0.97umol/L in the 1st group and from 0.49umol/L to 0.90umol/L in the 2nd group. Similar improvements were found in retinol binding protein viz. 21.28 mg/L to 31.06 mg/L in the 1st group and 17.05 mg/L to 24.80 mg/L in the 2nd group. At 8 weeks there was also no significant difference between the two groups either for serum retinol (0.69umol/L and 0.73umol/L respectively) nor for MRDR ratios (0.036 and 0.049 respectively). The MRDR results at 8 weeks indicated that these children did not have depleted vitamin A liver stores and that the low serum retinol levels seen at baseline were probably due to the acute phase response during an infectious episode. The results of these analyses showed no significant difference between the two treatment groups thus indicating that there was no benefit to giving vitamin A on recovery from an infectious episode instead of on admission, as is currently practised. / Thesis (M.Med.Sc.)-University of Natal, Durban, 2001.

Vitamin B-6 is a vitamin often promoted by the popular press as a cure all. It's role is also being studied in regard to pre-menstrual syndrome, myocardial infarction and alterations in lipid and fatty acid metabolism. This study was designed to investigate whether there was a difference between vitamin B-6 blood levels, during a baseline study, a period of vitamin B-6 depletion and vitamin B-6 supplemention in ten men ages 21-37. The effect of each diet on total cholesterol was also investigated.The experimentally accessible population for this study Laboratory as part of a larger study conducted by Dr. Stephen Coburn of the Fort Wayne State Developmental Center.Analysis of the data indicated:1) A significant difference between red blood cell pyridoxal phosphate and blood plasma levels of vitamin B-6 during the baseline, depletion and supplementation phases in ten men 21-37.2) Total serum cholesterol levels fell significantly through all phases of the study. High density lipoproteins fell significantly during the depletion phase but did not rise significantly during the supplementation phase. Low density lipoproteins showed no significant difference during the three phases of the study. When dietary records were evaluated mean dietary intake during the baseline and supplementation phases of the diet met the Recommended Dietary Allowance (RDA) for vitamin B-6. Mean protein intake also met the RDA with 102.1 grams during the baseline phase and 106.1 grams during the supplementation phase. These intakes are consistent with those found in previous studies conducted by the USDA. Mean intake of fat was lower than the 30 percent of calories recommended by the American Heart Association but wide variation existed among subjects.No physical symptoms of vitamin B-6 deficiency manifested themselves during the study. Subjects reported no other problems associated with low vitamin B-6 intakes.The data indicated that vitamin B-6 intake effects the amount of red blood cell plasma pyridoxal phosphate and plasma vitamin B-6. No clear effect can be found between vitamin B-6 intake and serum cholesterol levels. "Normal" diets also appeared to provide adequate vitamin B-6 to meet both RDA's and somatic needs. Wide variation seems to exist, however, among individuals. / Department of Home Economics