published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

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Circulating concentrations of vitamin D metabolites are associated with risk for a variety of diseases, including colorectal cancer. It is not known what level of circulating 25(OH)D is optimal for health; however, over-the-counter (OTC) vitamin D supplements are commonly used to improve status, though their effectiveness is unknown. It is also not known if polymorphic variation in genes associated with the vitamin D endocrine system is associated with differences in vitamin D metabolite levels or colorectal neoplasia.METHODS: A double-blind, randomized, placebo-controlled trial examined the effect of 400 IU OTC cholecalciferol on circulating concentrations of 25(OH)D. Associations between polymorphic variation in VDR, RXRA, GC, and CASR and circulating vitamin D metabolites or colorectal neoplasia were examined through analysis of the Ursodeoxycholic Acid (UDCA) and Wheat Bran Fiber (WBF) clinical trial data. A single nucleotide polymorphism (SNP) tagging approach was employed and a total of 42 VDR, 32 RXRA, 35 CASR and 25 GC tagSNPs were analyzed.RESULTS: The net change in serum 25(OH)D in the supplement versus placebo group was 2.3 ng/ml (8.5% change, P = 0.06). Principal components analyses revealed gene-level associations between RXRA and serum 1,25(OH)2D concentrations (p = 0.01) as well as GC and 25(OH)D concentrations (p < 0.01). Seven individual GC polymorphisms were significantly associated with circulating measures of 25(OH)D in addition to CASR polymorphism rs1042636 and proximal colorectal neoplasia (p-value =0.02), following a multiple comparisons adjustment. The CART analysis identified rs17467825 as predictive of continuous measures of 25(OH)D. GC polymorphisms rs1555563, rs7041, and rs222029 were identified as significantly predictive of the 25 ng/ml threshold for insufficiency.CONCLUSION: The results demonstrate that daily 400 IU OTC cholecalciferol is sufficient to maintain baseline concentrations of 25(OH)D in healthy adults, but not to significantly increase levels in all individuals. The results also identified polymorphisms in RXRA, GC, and CASR associated with or that predict vitamin D metabolite levels or colorectal neoplasia risk. The results justify further investigation on the optimal vitamin D supplementation dose for the general population and genetic variation that may be related to circulating concentrations of vitamin D metabolites or colorectal neoplasia.

Involvement of Cellular Retinoic Acid (CRABP) or Retinol (CRBP) Binding Proteins and 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) receptors in the response of cultured cells to retinoic acid and 1,25(OH)₂D₃ was examined. A new method for saturation analysis of CRABP and CRBP was applied to human tumors, human neuroblastoma cells, which retinoic acid causes to differentiate, and a bioselected subline resistant to retinoic acid. These data suggest that CRABP may not mediate cell differentiation by retinoic acid. In other studies, 1,25(OH)₂D₃ receptors and bioresponses were characterized in cultured primate cells. Rhesus monkey kidney cells (LLC-MK₂) were resistant to 1,25(OH)₂D₃-dependent induction of 25(OH)D-24-hydroxylase enzyme. The ED₅₀ in LLC-MK₂ cells was 10-100 fold higher than in other cultured cells. This resistance resulted from a low affinity receptor. Since the LLC-MK₂ variant receptor did not differ in size from the wild type rhesus 1,25(OH)₂D₃ receptor, (Mᵣ = 52 kDa) a subtle alteration in the receptor likely caused the decreased ligand affinity. Also of interest was the possible cellular resistance to 1,25(OH)₂D₃, in the owl monkey (Aotus trivurgatus), which generally occurs in new world primates. Owl monkey kidney (OMK) cells had the same content of receptors for 1,25(OH)₂D₃ and sensitivity to this hormone as cells from the rhesus monkey (old world primate). The ED₅₀ for induction of 24hydroxylase was 2-3 nM in both the OMK cells and the rhesus monkey fibroblasts. Both cells contained 2300 high affinity receptor molecules per cell, which bound DNA and were characterized by immunoblot as 52 kDa proteins. 1,25(OH)₂D₃ treatment increased the content of 1,25(OH)₂D₃ receptors in OMK cells, by increasing the synthesis of receptor mRNA. These data indicate the owl monkey is not resistant to 1,25(OH)₂D₃, unlike other new world primates. This finding was confirmed independently by demonstration that the owl monkey maintained mean serum 1,25(OH)₂D₃ levels (29 pg/ml) in the range of old world primates (33 pg/ml) and humans, in contrast to the elevated 1,25(OH)₂D₃ in other new world primates (97-129 pg/ml). This result suggests the alteration of 1,25(OH)₂D₃-endocrine dynamics in new world primates occurred subsequent to the evolutionary divergence of the owl monkey.

Bakgrund Frekvensenkät (FFQ) och kostregistrering är två vanliga kostundersökningsmetoder. För varje metod finns det felkällor och idag finns ingen fulländad kostundersökningsmetod. Genom att undersöka skillnader mellan kostundersökningsmetoder ökar förståelsen för att metoderna kan ge olika resultat. Vid kostundersökningar då näringsintag som t.ex. intag av vitamin D studeras, är det viktigt att ta hänsyn till energiintag. Detta för att ett missvisande energiintag kan ge ett felaktigt näringsintag. Syfte Syftet med studien var att jämföra eventuella skillnader mellan FFQ’s och kostregistreringar där barns intag av energi och vitamin D studerats. Metod FFQ där intaget av livsmedel uppskattades per månad, vecka eller dag och en vägd kostregistrering under tre dagar användes för att undersöka barns intag av energi och vitamin D. Barnen var mellan 5-8 år. Resultaten från de två metoderna jämfördes med varandra. Data analyserades i SPSS 22.0. Wilcoxon signed-rank test och correlate bivariate Spearman användes för att hitta skillnader respektive samband mellan resultaten från metoderna. Resultat Tjugosju FFQ’s jämfördes med 27 kostregistreringar. Medelåldern för barnen var 6 år och fyra månader. Det fanns en skillnad från uppskattningen av det totala intaget av vitamin D mellan de olika metoderna (p=0,001). Medelvärdet av intaget vitamin D från FFQ’s var 7,4 μg och 5,2 μg från kostregistreringarna. Enligt FFQ’s nådde sex barn det rekommenderade intaget (10 μg) av vitamin D. Inget barn nådde rekommendationen enligt kostregistreringarna. Intaget av fet fisk respektive intaget av mjölk 3% uppskattades i två fall lika mellan båda metoderna. Hos övriga deltagare skiljde sig intaget av alla livsmedel mellan kostundersökningsmetoderna. Slutsats Undersökningen visade signifikanta skillnader i resultaten av barns intag av vitamin D mellan FFQ’s och kostregistreringarna. Av den anledningen konkluderar vi att valet av kostundersökningsmetod påverkar resultatet. Eftersom FFQ´s visar ett medelintag över en längre tid kunde FFQ´s i den här studien med fördel använts som enda undersökningsmetod.

Funktionelle Analyse des Polymorphismus im Promotor des 24-Hydroxylase-Gens Ziel dieser Arbeit war es, in der Arbeitsgruppe eindeutig nachgewiesene Polymorphismen im Promotor des 24Hydroxylasegens, auf eine funktionelle Relevanz zu untersuchen. Die Substrate des Enzyms CYP24 sind 1,25(OH)2D3 und 25(OH)D3. Die entsprechenden Produkte der Katalyse sind 1,24,25(OH)3D3 und 24,25(OH)2D3. Über das erste Produkt wird das hochpotente 1,25(OH)2D3 im Sinne einer negativen Rückkopplung abgebaut. Dem zweiten Produkt 24,25(OH)2D3, konnte die Funktion als Botenstoff in der Knochenheilung und -entstehung nachgewiesen werden. Detailliertere Ergebnisse gibt es insbesondere für Chondrozyten in der Ruhezone der Epiphysefuge. Der Promotor des CYP24-Gens ist mit zwei Vitamin D-responsiven Elementen (VDREs) ausgestattet, welche die Transkriptionsrate des Gens in Anwesenheit von 1,25(OH)2D3 schnell und deutlich steigern. In einer poly-A-Strecke, die 488bp upstream des Transkriptionsstarts auffiel, konnte ein Polymorphismus nachgewiesen werden, die häufigsten Allele wurden als +A, +2A, und +C5AC bezeichnet. Mittels PCR wurden aus humaner, genomischer DNA, 672-680bp lange Promotorfragmente mit TATA-box, den beiden bekannten VDREs und dem polymorphen Bereich hergestellt. Diese Promotorfragmente wurden in den pGL3 Basic Vektor (ein für Luziferase kodierendes Plasmid ohne Promotor) kloniert und die Sequenz dieser Vektor-Promotorkonstrukte durch Fragmentanalyse und Sequenzierung kontrolliert. Die Vektor-Promotorkonstrukte wurden dann mittels Elektroporation in hFOB und T/C28 Zellen transfiziert. Es wurden Versuchsreihen unter basalen Bedingungen und unter Stimulation mit1,25(OH)2D3 durchgeführt. Anhand von Kontrollkonstrukten konnte die Spezifität der Promotoraktivität gezeigt werden. Für die Promotoren, die das Allel +A enthielten, konnte eine Verdoppelung und für die mit dem Allel +2A eine Verdreifachung der Luziferaseaktivität gezeigt werden. Das Allel +C5AC wies ähnliche Promotoraktivitäten wie der Wildtyp auf. Unter Stimulation mit 1,25(OH)2D3 war in allen Konstrukten mit einem Promotor in korrekter Orientierung eine mindestens dreifache Steigerung der Luziferaseaktivität zu messen. Sowohl Abwesenheit als auch in Anwesenheit von 1,25(OH)2D3 wird die Aktivität des Promotors des CYP24-Gens durch die Allele +A und +2A signifikant (p<0,001) gesteigert. Demzufolge könnte der Polymorphismus auch unter physiologischen Bedingungen Einfluss auf die Transkriptionsrate des CYP24-Gens haben. Da die CYP24-Aktivität vor allem über die Transkription reguliert wird, müsste durch den beschriebenen Polymorphismus die Aktivität des Enzyms in vivo gesteigert werden. In Folge könnte es lokal oder systemisch zu niedrigeren 1,25(OH)2D3- oder erhöhten 24,25(OH)2D3-Spiegeln kommen. Potentielle Bedeutung hat dieser Befund in der Pathogenese von Osteoporose, dem Prostatakarzinom oder anderen Erkrankungen die mit veränderten 1,25(OH)2D3 Aktivitäten einhergehen. Gegenwärtig wird bereits nach SNPs gefahndet die mit dem beschriebenen Polymorphismus assoziiert sind, um Untersuchungen von größeren Kollektiven auf diesen Polymorphismus hin zu erleichtern. Bell et al. konnten Veränderungen von CYP24-aktivitäten in Fibroblastenkulturen in Abhängigkeit von ethnischer Herkunft zeigen. Möglicherweise könnte das CYP24-Gen eines von zahlreichen Kandidatengenen sein, die bei der Entstehung von Osteoporose von Relevanz sind, und in ein System zur Risikoanalyse mit einfließen. Es könnten zum Beispiel über den Einsatz von zu entwickelnden „Single Nucleotid Polymorphims Gen-Chips“ solche Risikoprofile relativ einfach erstellt werden. In Folge könnten sowohl in der Prophylaxe als auch in der Therapie von Osteoporose neue Möglichkeiten geschaffen und neue Perspektiven eröffnen werden. / Functional analysis of the polymorphism in the promotor of the 24-Hydroxylase gene Aim: A functional characterisation of the Polymorphism in the promotor of the 24-hydroxylase (CYP24) gene. The enzyme CYP24 initiates the degradation of the active vitamin D metabolite 1,25(OH)D3 as well as catalysing the production of 24,25(OH)D3, an active metabolite bone metabolism. If the polymorphism influenced the activity of CYP24, local or systemic levels of vitamin D could be altered or bone metabolism disturbed by varying 24,25(OH)D3 levels. The polymorphism could a factor in the pathogenesis of osteoporosis or other diseases of the skeletal system. Methods: Four different alleles of the polymorphism in the promotor of the CYP24 gene (WT, +A, +2A and +C5AC) as well as controls were amplified using PCR and cloned into a luciferase assay vector (pgl3-basic). Using electroporation the vector was transfected into two different cell systems, h-fob cells (human Osteoblasts) and T/C28a2 cells (human Chondrocytes). The cells were cultivated with and without the influence of vitamin D. The promotor activity was quantified using the luciferase assay. Results: By comparison with the controls a specific promotor activity could be demonstrated. For the allele +A a two fold increase and for the allele +2A a three fold increase in promotor activity compared to the wild type promotor was measured (p<0,001). The allele +C5AC showed no significant influence on the promotor activity. Under influence of vitamin D promotor activity was increase at least three times as high as in the basic setting. Discussion: Since the activity of the enzyme CYP24 is controlled mainly by the rate of transcription, the polymorphism in the promotor of the gene could influence CYP24 activity in vivo. The result could be a local or systemic decrease of 1,25(OH)D3 levels or increase of 24,25(OH)D3 levels. The polymorphism might therefore be relevant in the pathogenesis of osteoporosis, prostate cancer or other diseases associated with decreased levels of vitamin D.

INTRODUCTION: In this study, we identified the association of Vitamin D with orthodontic tooth movement and the impact of Vitamin D 1,25OHD and 25OHD forms on osteoblast function. MATERIALS AND METHODS: This study is comprised of two parts; a clinical and a laboratory part. In part I, saliva samples were collected from orthodontic patients each month for the first six months of orthodontic treatment along with casts at the beginning and the end of the study period. The samples were measured for Vitamin D binding protein (VitDBP) and alkaline phosphatase (ALP) and correlated with clinical tooth movement using absolute change in irregularity index (II). In part II, osteoblasts were collected from the calvaria of 3-5 day old healthy wild-type mice and cultured with differing concentrations of 1,25OHD (1, 10 and 100nmol) and 25OHD (100, 200, 400 nmol). ALP, OPG, and RANKL were measured as outcomes of Vitamin D treatment of osteoblasts. Intracellular signaling in response to Vitamin D was assessed by identifying the phosphorylation of ERK 1/2, p38 and NLK in primary osteoblasts. RESULTS: Measurement of salivary Vitamin D binding protein (VitDBP) showed that both low (<2.75 ng/ml) and high (>6.48 ng/ml) logVitDBP were associated with reduced tooth movement. There was no significant correlation between ALP levels and orthodontic treatment. Significant seasonal changes in VitDBP using a two-season year model were found with lower levels noticed in the summer (Mar-Sept) than in the winter (Oct-Feb) at p<0.05. A decrease in OPG production with higher concentrations of 1,25OHD and 25OHD with a corresponding increase in RANKL levels in primary osteoblast cultures was found. Similar to the clinical findings, ALP levels were not significantly affected by increasing concentrations of both 1,25OHD and 25OHD. The ERK 1/2 showed upregulation in response to treatment with 1,25OHD and downregulation in response to treatment with 25OHD concentrations. Meanwhile, p38 and NLK were affected by 1,25OHD and not by 25OHD. CONCLUSIONS: Clinical outcomes of orthodontic treatment are associated with a range of optimal Vitamin D binding protein (VitDBP) as detected in saliva. Different forms of Vitamin D affect osteoblast response and signaling differently.

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Master of Medicine in the branch of Anaesthesiology / Background and Objective There has been a recent resurgence of interest in vitamin D and its far-reaching effects in physiology and pathophysiology. Theatre personnel, and all indoor workers, should be cognisant of vitamin D deficiency as a real occupational hazard. Vitamin D deficiency is a global problem that has been studied extensively in colder climates and even been found in warmer climates. No research was identified among medical personnel in South Africa. The primary objective of this study was to describe serum 25-hydroxyvitamin D (25(OH)D) levels of anaesthetists. The secondary objective was to describe and compare factors influencing vitamin D levels in anaesthetists who are vitamin D insufficient to those who are not. These factors included: ethnicity, gender, body mass index (BMI), multivitamin use, calcium or vitamin D supplementation, sun exposure, vitamin D intake from diet alone, vitamin D intake from diet and supplementation and calcium intake (dairy). Methods Data was collected over a period of one month, in winter (mid-July to mid-August 2013). On the morning of sample collection anaesthetists agreeing to participate signed the informed consent (Appendix 2), prior to enrolment in the study. The anaesthetists then completed the questionnaire (Appendix 5). The following data was obtained from the questionnaire: age, gender, ethnic group, dietary supplementation, sun exposure, sunscreen use, BMI and diet. Each participant had 5 ml of blood collected in a standardised manner into a purple top ethylenediaminetetraacetic acid blood specimen tube. The processing of samples was done by qualified laboratory personnel using standard chemical pathology equipment and procedures. High Performance Liquid Chromatography was performed to determine 25(OH)D levels using a Shimadzu® Nexera X2 Ultra performance liquid chromatography system with a photodiode array detector (Shimadzu®, Japan). Results The median 25(OH)D was 43.8 nmol/l (IQR 26-76), with 51 of 89 (57.30 %) anaesthetists being vitamin D insufficient. There was a statistically significant association between ethnicity and vitamin D status (p<0.001). Twenty-one (80.77 %) Indian anaesthetists and 14 (70.00 %) black anaesthetists were vitamin D insufficient, as compared to only 10 (28.57%) white anaesthetists. There was no significant association between the other secondary objectives-gender (p=0.60), sun exposure (p = 0.93), vitamin D intake from diet alone (p= 0.07), vitamin D intake from diet and supplementation (p=0.05) and calcium intake (p=0.55) and vitamin D status. There was no significant difference between BMI and vitamin D status. When a comparison was made between the two groups of BMI <25 and BMI ≥25, using a Mann-Whitney test the two-tailed P value was 0.6791. There was a significant association between multivitamin use (p=0.01) and vitamin D status. Conclusion Vitamin D should no longer be a forgotten vitamin. The insufficient vitamin D levels of anaesthetist in this study, puts them at risk for pathology far beyond bone health. Adequate vitamin D levels should be seen as essential, rather than optional, even in “sunny” climates. / MT2016

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.

Acute respiratory infections (ARI) are a major global cause of morbidity and mortality. Vitamin D deficiency has been reported to associate with susceptibility to ARI and with greater severity and poorer control of asthma and chronic obstructive pulmonary disease (COPD). Clinical trials of vitamin D for the prevention of ARI have yielded heterogeneous results, with some showing protection and others not. This may reflect variation in the frequency of genetic variants influencing response to vitamin D supplementation in different populations. The impact that genetic variation in the vitamin D pathway has on vitamin D status, disease phenotype and response to vitamin D supplementation in prevention of ARI has not been comprehensively investigated. Methods: I conducted: 1. A systematic review and meta-analysis of clinical studies which have investigated vitamin D as a potential therapy for ARI; 2. Three cross-sectional studies (in n=297 adult asthma patients, n=278 COPD patients, and n=272 older adults) to investigate potential environmental determinants (lifestyle and anthropometric) and genetic determinants (35 single nucleotide polymorphisms [SNP] in 11 vitamin D related genes) of serum 25-hydroxyvitamin D concentration (25[OH]D) and clinical phenotype; 3. Three prospective studies investigating the influence of genetic variation in the vitamin D pathway on a) susceptibility to ARI (main effects analysis) and b) efficacy of vitamin D supplementation for the prevention of ARI (interaction analysis). Results: My systematic review identified consistent reports of an inverse association between vitamin D status and risk of ARI in observational studies, and heterogeneous reports from clinical trials. My cross-sectional studies identified a range of classical environmental factors which predict vitamin D status in the three study populations, but did not identify any genetic variants in the vitamin D pathway that associate with vitamin D status. I identified an association between vitamin D deficiency and decreased lung function in COPD patients, but no associations between vitamin D deficiency and asthma phenotype. Finally, my analysis identified a haplotype of 5 single nucleotide polymorphisms in the vitamin D receptor (VDR) gene which significantly modify the effect of vitamin D supplementation on risk of upper respiratory infection in COPD patients. Conclusions: I identified environmental determinants that predict 25(OH)D concentrations in all three study populations, but only found an association between vitamin D deficiency and disease severity in COPD patients. Furthermore, I identified a haplotype in VDR which modifies the effect of vitamin D supplementation in COPD patients to result in a significantly reduced risk of ARI.