The kidney represents a unique endocrine organ for the metabolism and action of vitamin D₃. Renal biosynthesis of 1,25-hydroxyvitamin D₃ (1,25(OH)₂D₃), and other vitamin D₃ metabolites, is regulated by ionic and hormonal factors. 1,25(OH)₂D₃ elicits a dramatic increase in 25-hydroxyvitamin D₃-24-hydroxylase (24-OHase) activity while concomitantly suppressing 25-hydroxyvitamin D₃-1α-hydroxylase (1-OHase) activity. To study this mechanism(s), biosynthesis and purification of the radiolabeled metabolites in question was necessary. Protocols for the selective induction of either 1-OHase or 24-OHase activity in chick kidney (in vivo) and the use of these kidneys (homogenates) for the biosynthesis of very high specific radioactivity metabolites, in vitro, are presented. A number of chromatographic systems including HPLC were developed and employed for the detection and purification of these biosynthetic metabolites (1,25(OH)₂[³H]D₃; 1,24,25(OH)₃[³H]D₃; 24,25(OH)₂[³H]D₃). Characterization of specific receptors for 1,25(OH)₂D₃ in the rat and chick kidney is presented and by comparison, these receptors appear similar to the chick intestinal component. 1,25(OH)₂D₃ receptors were also identified in a number of cultured mammalian cells. These receptors, irrespective of origin (tissue or species), selectively bind to immobilized DNA, a property which may indicate a physiologic function. Extensive metabolite binding studies indicate that 1,24,25(OH)₃D₃ is the second most active metabolite at the receptor level. To evaluate the role of the 1,25(OH)₂D₃ receptor in modulating the 1,25(OH)₂D₃-responsive activity of the renal 24-OHase enzyme, two renal cell lines (LLC-PK₁ and LLC-MK₂) were studied. A sensitive microassay for 24-OHase activity in intact cells was developed to monitor the enzyme responses. Although both cell lines contain 1,25(OH)₂D₃-responsive 24-OHase activity, only the LLC-PK₁ cells contain receptors for 1,25(OH)₂D₃. It is concluded that 1,25(OH)₂D₃ can rapidly and specifically regulate (induce) the activity of the 24-OHase enzyme in the absence of its specific receptor protein and this induction is inhibited by actinomycin D. Hence, although 1,25(OH)₂D₃ may act in renal cells to induce enzymes for the metabolism of vitamin D via its receptor component, this mechanism is not obligatory.

Thesis (M.Sc.). / Written for the School of Dietetics and Human Nutrition. Title from title page of PDF (viewed 2008/12/09). Includes bibliographical references.

Thesis (Ph.D.) in Food and Nutrition Sciences--University of Maine, 2007. / Includes vita. Includes bibliographical references (leaves 133-152).

No description available.

In humans, the epidermis is the main site for the synthesis of Vitamin D3 (cholecalciferol) from 7-dehydrocholesterol. Cholecalciferol undergoes further hydroxylation in the liver and kidney to produce the active form of the circulating hormone 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). In target cells, 1,25(OH)2D3 interacts with the specific intracellular vitamin D receptor (VDR), a member of the nuclear receptor superfamily. However, epidermal keratinocytes, in addition to being target cells, have enzymes required for autocrine production of 1,25(OH)2D3. They can convert cholecalciferol to 1,25(OH)2D3 via 25-hydroxylase (CYP2R1) and 1α-hydroxylase (CYP27B1). Another enzyme, 24-hydroxylase (CYP24A1), regulates local levels by inactivating 1,25(OH)2D3. While recent studies have shown that absence of VDR or 1,25(OH)2D3 impairs formation of granulation tissue during wound healing in mice, little is known about the autocrine and paracrine regulation of biologically active vitamin D3 by human dermal fibroblasts during cutaneous wound healing. Primary cultures of human keratinocytes and fibroblasts expressed VDR and all the cytochrome enzymes necessary for autocrine production of vitamin D. The relative expression of VDR mRNA was higher in dermal fibroblasts than donor-matched keratinocytes. In contrast, epidermal keratinocytes had a higher mRNA expression of vitamin D3 metabolising enzymes. A scratch wound assay confirmed that 1,25(OH)2D3 stimulated keratinocyte migration, but paradoxically inhibited fibroblast migration as early as 4h, yet neither cholecalciferol nor 25-hydroxyvitamin D3 had any effect. VDR knockdown using small interfering RNA (siRNA) abolished the inhibitory effect of 1,25(OH)2D3 on fibroblast migration, demonstrating the requirement for the VDR in this response. Immunofluorescent staining revealed that 1,25(OH)2D3 increased nuclear VDR protein expression, without a corresponding increase in VDR mRNA transcription only in mechanically wounded dermal fibroblasts, indicating activation of the receptors. Incubation with either 1,25(OH)2D3, cholecalciferol or 25(OH)D3 up-regulated CYP24A1 transcription. This response was most pronounced with 1,25(OH)2D3, suggesting a tightly regulated feedback control on 1,25(OH)2D3 bioavailability within the dermis. In addition, cholecalciferol also increased CYP2R1 and CYP27B1 mRNA expression in scratched dermal fibroblasts, providing evidence for autocrine regulation of 1,25(OH)2D3 by dermal fibroblasts. Expression of α-SMA protein was up-regulated in cultured dermal fibroblasts following scratching, which was down-regulated in the presence of 1,25(OH)2D3. These observations suggest that 1,25(OH)2D3 may restrict differentiation of wounded dermal fibroblasts into pro-fibrotic myofibroblasts. 1,25(OH)2D3 also down-regulated MMP-2 secretion and collagen type I to III ratio in scratched dermal fibroblasts. Using a human ex vivo wound healing model, it was demonstrated that 1,25(OH)2D3, but not cholecalciferol, stimulated the rate of wound closure. In summary, this study has confirmed that human dermal fibroblasts express the transcriptional machinery for autocrine production of 1,25(OH)2D3, and a higher VDR expression suggests they are more responsive than keratinocytes. Changes in CYP and VDR expression in the presence of cholecalciferol, 25-hydroxyvitamin D3 or 1,25(OH)2D3 indicate fine-tuning of the bioavailability of vitamin D in the dermis after wounding. Down-regulation of α-SMA, MMP-2 secretion and the collagen type I to III ratio by 1,25(OH)2D3 highlight an important role for 1,25(OH)2D3 in modulating wound healing and the scarring process.

There is discordance in the vitamin D literature between correlational studies, which show an association between higher vitamin D exposure and good health, and randomized controlled trials of vitamin D supplementation, which are inconclusive. I test the theory that this discordance is due to false assumptions about how vitamin D affects human health. I use the method of systematic review and meta-analysis—accepting only experimental studies that supplement with the animal version of vitamin D, D3, not D2 or vitamin D metabolites or analogues, as well as accepting only studies with daily rather than less-frequent but larger doses—to show that daily vitamin D3 supplementation has a statistically-significant beneficial effect on blood pressure and markers of diabetes. Using nationally-representative correlational data, I also demonstrate that the health disparities in blood pressure, if not diabetes, will be eliminated only with new health policies dedicated to health education on the vast nutritional difference in vitamin D status between black and white Americans. As a part of this dissertation, I also developed an online multi-user web application to facilitate systematic reviews and meta-analyses.

Vitamin D3 (cholecalciferol) (Vit D3) is a form of vitamin D (Vit D) that is essential for normal body function in humans. Vit D is a fat-soluble vitamin used to prevent osteoporosis in adults, prevent rickets in children, and supplement Vit D deficiencies which are seen worldwide. Physicians usually recommend 2,000 International Units (IU) Vit D3 for these deficiencies, but due to the poor aqueous solubility of Vit D3, this dose can be insufficient for patients. It would be more beneficial to recommend 5,000 IU. In 1994, the Dietary Supplement Health and Education Act was passed defining vitamins as “dietary supplements” and no longer required FDA registration prior to marketing. Dissolution results are expressed as the percent of the content released into solution, with a target of 85 to 125%. There is not an effective dissolution test existing for Vit D3 product evaluation, so an optimized dissolution test for Vit D3 was developed and used to evaluate non-prescription Vit D3 products. The current FDA guideline for Vit D3 dissolution is USP Apparatus II with 500 mL of 0.3% sodium dodecyl sulfate (SDS) in water. Since Vit D3 is not very water soluble, a surfactant is required for it to go into solution. The research was started with a larger volume (1 L) and with commonly used pharmaceutical grade surfactants: Triton X-100, Tween 80, Span 20, and sodium lauryl sulfate (SLS). SDS was eliminated due to interference with the HPLC analysis. Using two Vit D3 test products, which previous research found to have consistent drug content, each of these surfactants were tested at 0.8%. After evaluating the HPLC results, Triton and Tween were eliminated due to too much interference with the analysis process. The interference consisted of a large initial solvent front peak, elevated baseline, overlap with the drug peak, and required significant decrease in the mobile phase flow rate. Changing the mobile phase to 50/50 methanol/1-propanol solved the flow rate issue and peak separation overlap, but not the large solvent peak or elevated baseline. Dissolution testing continued using SLS and Span using 0.6%. Dissolution of the two test products, designated A and B, with 2 representing 2000 IU and 5 being 5000 IU, yielded higher values using SLS (A2 75.5%; A5 152.8%; B2 30.9%; B5 36.6%) compared to Span (A2 15%; A5 136%; B2 20.3%; B5 63%). Additional work continued to determine the best concentration of SLS. Better dissolution results were obtained using 1.2% (A2 88.1%; A5 156.7%) when compared to 0.3% (A2 87.4%; A5 153.2%), 0.6% (A2 70.7%; A5 132.2%), and 0.9% (A2 79.7%; A5 150.5%). Use of 1.5% did not yield a significant improvement (A2 84% vs 86.8%; A5 146.4% vs 144.6%), so 1.2% was chosen as this would minimize the amount of surfactant in the samples. The final dissolution method was created using a USP Apparatus II in 1000 mL containing 1.2% SLS in water. Based upon these characteristics, SLS 1.2% gave a good balance of minimizing surfactant concentration and analytical interference while maximizing Vit D3 dissolution.

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.

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.

Primarily synthesised via cutaneous exposure to solar ultraviolet B (UVB) radiation, serum vitamin D concentrations, measured as 25-hydroxyvitamin D (25(OH)D), fluctuate according to solar availability. Seasonal variations in vitamin D are common in areas of high or low latitude determined by the distance from the sun. Seasonal variations in blood pressure, immune markers and some diseases including influenza, have also been reported. However, the contributions of UVB light or vitamin D on the immune markers have not been fully determined. Against this background, the purpose of this research was to investigate the effects of UVB light therapy and dietary vitamin D supplementation on markers of immune function. The D SIRe1 study aimed to assess whether dietary-derived 25(OH)D could have similar effects on immune function as light-derived 25(OH)D. The study was an 8-week comparative intervention trial in healthy adults randomised to receive either 3 times weekly UVB radiation (equivalent to doses received during a Grampian-summer) for 4 weeks; or oral vitamin D3 (1000 IU a day for 8 weeks). Total 25(OH)D was measured by dual tandem mass spectrometry of serum samples following removal of protein and de-lipidation, whilst regulatory T cells (Tregs), known for maintaining immune system homeostasis, by flow cytometry. The study showed similar short-term effects between oral vitamin D and UVB exposure on measured outcomes. However, study interpretation was limited by the lack of a placebo group, yet, to our knowledge, this was the first study to directly compare dose-matched UVB therapy and vitamin D supplementation in healthy participants. Using similar laboratory techniques, the D-SIRe2 study, a placebo-controlled trial, assessed short-term (12 weeks) and long-term (43 weeks) effects of vitamin D supplementation on immune markers. Commencing in spring (March) and finishing in winter (January) 2015/2016, the study showed seasonal fluctuations in most immune markers. The fluctuations did not change according to variations in 25(OH)D concentrations nor were they correlated with solar UVB doses, with the exception of T cell proliferative responses, which were positively correlated with daily solar UVB doses. An interesting finding from this study was the prevention of increases in pro-inflammatory IFN-γ cytokine concentrations in the spring and summer time in the vitamin D3 supplemental group versus placebo. IFN-γ concentrations were raised from 7940 pg/mL at baseline in March, to roughly 12400 pg/mL at week 4 and to 13909 pg/mL at week 12 in the placebo group. The concentrations were roughly 1.3 times the mean concentrations measured in the vitamin D group at the timepoints following baseline concentrations of 10678 pg/mL, and 10013 pg/mL and 10233 pg/mL at weeks 4 and 12, respectively. The interactions between solar light or seasonal effects and oral vitamin D supplementation, as well as their individual and combined effects on immune function, are yet to be fully determined. Moreover, the metabolic and physiological implications of seasonal variation in serum 25(OH)D concentration and markers of immune function are currently unknown, requiring further investigation.