Philosophiae Doctor - PhD / Background: A subtype of inflammatory bowel disease, Crohn' s disease is thought to represent a complex interaction between environmental factors, a defective immune system, the gastrointestinal microbiome and genetic' susceptibility; however; the-prevalence of different susceptibility mutations appears to vary between population groups, implying distinctions in disease pathogenesis or risk. Vitamin D, signaling through the vitamin D receptor, appears to have numerous effects on the immune system, and deficiency has been shown to playa role in both the pathogenesis and severity of experimental inflammatory bowel disease. However, the literature surrounding the association between vitamin D concentrations and disease severity in Crohn's disease is limited, and no such literature exists in South Africa. Furthermore, a paucity of data exists on the racial variability of Crohn' s disease phenotype in the Western Cape population of South Africa, as well as environmental factors in childhood associated with future Crohn's disease development. Aims: The three primary aims of the study were to investigate: 1) the racial variability of, Crohn's disease phenotype, defined by the Montreal classification scheme, as well as Crohn's disease behavior, using predefined definitions, stratified as 'complicated' or 'uncomplicated', based on a cross-sectional study design; 2) the association between childhood environmental exposures and the subsequent development of Crohn's disease, with specific emphasis on the timing of exposure, based on a case-control study design; and 3) the association between serum 25(OH)D concentration with Crohn's disease activity, measured by the Harvey Bradshaw Index, based on a cross-sectional study design; in this process, various vitamin D thresholds for predicting a high disease activity score were investigated, and the serum 25(OH)D concentrations were compared with those of the healthy controls to evaluate the prevalence of vitamin D deficiency. Design: This was a case control study, as well as two cross-sectional evaluations of the case control study data, of all consecutive Crohn's disease patients (ages 18-70 years) seen between September 2011 and January 2013 during their normally scheduled appointments at Schuur Hospital and Tygerberg Hospital. Control subjects for the study were identified from the same populations giving rise to the Crohn's disease cases. An investigator-administrated questionnaire was used to identify numerous demographic and lifestyle variables, as well as childhood environmental exposures during three age intervals; 0-5, 6-10 and 11-18 years. Clinical variables at diagnosis and time of study enrolment were determined via a review of medical and pharmacy records, as well as clinical examination by the consulting gastroenterologist. Serum 25(OH)D was measured using the SIEMENS ADIVA Centaur® XP Vitamin D Immunoassay [Siemens Healthcare Diagnostics Inc., Tarrytown, NY, USA]. Vitamin D status was classified as either 'deficient' or 'sufficient', and was analyzed in 2 ways: ~20 ng/mL versus ~21 ng/mL; and ~29 ng/mL versus ~30 ng/mL, respectively. One year after study completion, a total of 40 (10%) randomly selected participants from the cohort completed the interviewer-administered questionnaire for a second time. A kappa statistic was used in order to measure the agreement between repeated data for the questionnaire. Only data pertaining to the three age intervals (0-5, 6-10 and 11-18 years) was extracted in this process. Results: One hundred and ninety four Crohn's disease patients and 213 controls meeting our inclusion criteria were identified; 35 (18%) and 19 (9%) were White, 152 (78%) and 177 (83%) were Coloured, and 7(4%) and 17 (8%) were South African Black, respectively. No subjects reported being of Asian or Indian ethnicity. Overall, 125 (31%) of the cohort were male. On multiple logistic regression analysis, Coloured Crohn's disease patients were significantly more likely to develop 'complicated' Crohn's disease (60% versus 9%, P = 0.023) during the disease course when compared to White Crohn's disease patients. In addition, significantly more White subjects had successfully discontinued cigarette smoking at study enrolment (31% versus 7% reduction, P = 0.02). No additional interracial differences were found. A low proportion inflammatory bowel diseases family history was observed among the Coloured and Black subjects. When evaluating childhood environmental exposures, multiple logistic regression analysis showed that during the age interval 6-10 years, never having consumed unpasteurized milk [(OR = 6.43; 95% Cl, 3.02-14.81), (K =0.79; 95% Cl, 0.39-1.00)] and never having a donkey, horse, sheep or cow on the property [(OR = 3.10; 95% Cl, 1.42-7.21), (K = 0.84; 95% Cl, 0.12-1.00)], significantly increased the risk of developing future Crohn's disease. During the age interval 11-18 years, an independent risk-association was identified for; never having consumed unpasteurized milk (OR = 2.60; 95% Cl, 1.17-6.10) and second-hand cigarette smoke exposure (OR = 1.93; 95% Cl, 1.13-3.35). For the vitamin Danalysis, 186 Crohn's disease patients and 199 control subjects met the study inclusion criteria. Overall, 113 (29%) of the cohort were male. Forty four percent of the cohort had a deficient vitamin D concentration (::;20 ng/ml.), no participants had severely deficient vitamin D concentrations, and 26% of the cohort had sufficient vitamin D concentrations (:::30 ng/mL). Fifty-three percent of the controls and 34% of the cases had vitamin D concentrations ::;20 ng/mL (P < 0.001). On multiple logistic regression analysis, higher Harvey Bradshaw Index scores and not having taken vitamin D supplementation in the six months prior to enrolment were identified as independent predictors of vitamin D deficiency in Crohn's disease patients; defined either as ::;20 ng/mL, or as ::;29 ng/mL (P < 0.001). Compared to patients with Harvey Bradshaw Index <5, those with Harvey Bradshaw Index 2:8 were 2.5-times more likely to have vitamin D concentrations ::;21 ng/mL (PR = 2.5; 95% Cl, 1.30-6.30). The risk was similar, though not as high, if deficiency was defined as ::;29ng/ml. (PR = 2.0; 95% Cl, 1.20-3.50). Conclusions: Coloured Crohn's disease patients were significantly more likely to develop 'complicated' Crohn's disease over time when compared to White Crohn's disease patients. Limited microbial exposures and exposure to second-hand cigarette smoke during childhood is associated with future development Crohn's diseases. However the inconsistencies between each age interval with regards to the identified risk factors may imply that the effect of different viruses or bacteria on the development of immune structures varies according to the timing of exposure. The finding that lower serum 25(OH)D was associated with moderate to severe Crohn's disease activity suggests that this patient population may benefit from vitamin D supplementation in order to achieve, or maintain a serum 25(OH)D concentration of at least 30 ng/mL.

Vitamin D insufficiency remains a costly pandemic in veterans. Treatment requires achievement of desired 25-hydroxyvitamin D [25(OH)D] concentrations. The frequency with which 25(OH)D should be measured following treatment remains speculative. A retrospective analysis of veterans with vitamin D insufficiency was conducted. The group was stratified on the basis of initial 25(OH)D and assessed for frequency of follow-up 25(OH)D concentrations. Over 3 years, 278 patients with insufficient 25(OH)D concentrations were identified. Of these, 87 (31%) patients had subsequent levels assessed in the year following initial documentation of vitamin D insufficiency. The likelihood of follow-up testing was unrelated to the initial vitamin D level. In the patients with follow-up 25(OH)D levels, 90% eventually achieved a serum level of 30 ng/mL or greater. Veterans with vitamin D insufficiency have inadequate serial monitoring of 25(OH)D concentrations.

Background: Traumatic brain injury (TBI) still remains a difficult disorder to treat. TBI has been associated to chronic neuroinflammation and a high risk for neurodegenerative disorders. Since 2001 between ten to twenty percent of all deployed military members have suffered a combat-related TBI. Nearly twenty to thirty percent of those will experience chronic cognitive, behavioral and somatic symptoms after suffering a TBI. Methods: The objective of this review is to evaluate current literature examining vitamin D as a neurosteroid with protective properties and its clinical relevance after traumatic brain injury. Vitamin D is known to participate in neurobiological processes and genomic regulation in the brain. Clinical and laboratory findings support that vitamin D modulates the immune responses to trauma, diminishes oxidative and toxic damage, and inhibiting activation and progression of the neuroinflammation. Inadequate levels of vitamin D have been identified as a common risk factor for many neurological disorders and have been linked to poorer recovery. Results: This review found compelling evidence to support that the pathology of TBI is closely associated with neuroprotective mechanisms of vitamin D. Low vitamin D levels are common among US active duty military and veterans. The findings strongly suggest that optimizing vitamin D prior to injury could improve the recovery for military members after experiencing a TBI. Vitamin D ameliorates brain damage by modulating neuroinflammation, improving cell survival and down-regulating mechanisms involved in the progression of cell damage following a TBI. However, further studies are needed to evaluate the effects of vitamin D optimization in TBI outcomes.

Aktuelle Studien legen nahe, dass ein niedriger Vitamin D-Status assoziiert mit steigenden Breitengraden, mit dem Auftreten von Autoantikörpern und damit einhergehenden Autoimmunerkrankungen positiv korreliert. Trifft UV-B-Strahlung auf die Haut, entsteht aus 7-Dehydrocholesterol nach enzymatischen Prozessen in Leber und Niere, die bioaktive Form von Vitamin D, 1α,25-Dihydroxyvitamin D3 (Calcitriol). Den finalen Stoffwechselschritt katalysiert die 1α-Hydroxylase CYP27B1. Die biologische Wirkung von Calcitriol wird über dessen Bindung an den nukleären Vitamin D-Rezeptor vermittelt, was letztlich zur Transkription der Zielgene führt. Die T-Zell-abhängige Sensibilisierung von Vitamin D-defizienten Cyp27b1-KO- und Wildtyp-Tieren mit Ovalbumin zeigte verstärkte humorale Immunantworten mit erhöhten Konzentrationen von Gesamt- sowie spezifischem IgG1, IgE und IgA bei den Cyp27b1-KO-Mäusen. Die Untersuchung der Leukopoese der Cyp27b1-KO-Tiere zeigte, dass die untersuchten lymphatischen Organe verminderte Gesamtzellzahlen gegenüber Wildtyp-Tieren aufwiesen. Die Präsenz und Verteilung in den jeweiligen Zellkompartimenten offenbarte keine wesentlichen Abweichungen zwischen Cyp27b1-KO- und Wildtyp-Mäusen. In einem Krankheitsmodell mit dem Helminthen Heligmosomoides polygyrus bakeri fielen die Cyp27b1-KO-Tiere durch dem Wildtyp gegenüber erhöhten Gesamt- sowie spezifischen IgE-Werten auf. Zwischen beiden Genotypen zeigten sich keine Unterschiede bei parasitologischen Parametern wie der Wurmlast, der Eianzahl pro Gramm Faeces sowie der Fekundität. Die Ergebnisse dieser Arbeit bestätigen, dass endogen-produziertes Vitamin D einen Einfluss auf die Funktionsweise von Lymphozyten hat. Dies äußert sich in verstärkten IgE-abhängigen Immunantworten bei Cyp27b1-KO-Tieren. In einem Parasiteninfektionsmodell wurden erneut verstärkte IgE-Antworten beobachtet, jedoch waren keine pathophysiologischen Konsequenzen in Bezug auf die Wurmabwehr nachweisbar. / Current studies demonstrate that low vitamin D levels associated with higher latitudes correlate with the occurrence of autoantibodies and linked diseases. Following UV-B radiation of the skin, numerous enzymatic reactions in liver and kidneys causes 7-dehydrocholesterol to turn into the bioactive 1α,25-dihydroxyvitamin D3 (calcitriol). The final and crucial step is thereby performed by the enzyme CYP27B1, an 1α-hydroxylase. The effect of calcitriol is mediated through binding to the vitamin D receptor, resulting in the transcription of target genes. T cell-dependent sensitization of Cyp27b1-wildtype and Cyp27b1-KO mice with ovalbumin revealed an increased humoral immune response in Cyp27b1-KO mice reflected by elevated concentrations of total and specific IgG1, IgE and IgA. Analysis of the leukopoiesis showed a diminished total cell count in bone marrow, thymus, spleen and peritoneal cavity in Cyp27b1-KO compared to Cyp27b1-wildtype mice. However, appearance and distribution of the analyzed cell compartments were comparable. A disease model using the intestinal nematode Heligmosomoides polygyrus bakeri demonstrated enhanced secretion of total and specific IgE in Cyp27b1-KO mice, which confirmed our previous findings. However, this showed no effect on parasite rejection, as seen in comparable results for worm burden, eggs per gram faeces and fecundity of female worms in Cyp27b1-wildtype and Cyp27b1-KO mice. Our work verified the role of endogenous vitamin D for lymphocyte development revealed by increased IgE-dependent immune responses in Cyp27b1-KO mice. Infection with H.p. bakeri confirmed enhanced IgE-responses, however, these results revealed no benefit in parasite clearance.

In the early 19th century a ground-breaking discovery was made that linked a dietary deficiency of a fat-soluble vitamin with the childhood disease known as rickets. The vitamin was named vitamin D and extensive research regarding the physiological importance of this vitamin followed ever since. It is currently known that vitamin D plays an important role in maintaining the calcium and phosphate homeostasis in the human body. Less clear evidence states the medical importance of vitamin D in the prevention and cancer, autoimmune disease and diabetes. Current literature shows that vitamin D has five distinct forms, vitamin D1 to D5, of which vitamin D2 and D3 are the most studied forms. The term “vitamin D” is often wrongfully used to include the vitamin D mother molecule, the vitamin D status indicator (25(OH)D), the biologically active form (1,25(OH)2D) and biologically inactive form (24,25(OH)2D). The interest for measurement of these vitamin D analogues is a continuously growing field both on individual and epidemiological level. For decades laboratories have struggled to produce a robust method capable of quantifying these different vitamin D analogues and uncovered a new form of complexity regarding the analysis of these analogues. The identification of the C3-epimeric forms of vitamin D metabolites has forced laboratories to rethink their analytical methods and several concerns were raised regarding the overestimation of the true vitamin D status by current analytical methods. The quantification of the biologically active and inactive forms of vitamin D is reported to be difficult and to date very few LC-MS/MS methods reported in the literature are able to quantify various vitamin D analogues. However, to our knowledge none of these methods are able to include the precursor vitamin D, the 25-hydroxylated metabolites, the biologically active and inactive metabolites, C3-epimers and isobaric compounds in a single run. Therefore the aim of this study was to develop, optimise and validate a LC-MS/MS method for the quantification of twelve vitamin D analogues in a single run. This was done by optimising the underlying LC-MS/MS parameters to ensure optimal analytical sensitivity in positive ESI mode and sufficient chromatographic separation between analytes with similar chemical properties. Furthermore, the optimised method was validated to ensure the accuracy and precision of the method before implementation into a clinical environment. The vitamin D analogues included in this study were vitamin D2, vitamin D3, 25(OH)D2, 25(OH)D3, 1,25(OH)2D2, 1,25(OH)2D3, 24,25(OH)2D2, 24,25(OH)2D3, 3-epi-25(OH)D2, 3-epi- 25(OH)D3, 7(OH)4C3 and 1α(OH)D3. A double liquid-liquid extraction with hexane and ethyl acetate were found to be the most efficient at extracting the vitamin D analogues from a serum matrix after matrix modification with sodium hydroxide. Recoveries of > 95 % (CV <10 %) were achieved for all the analytes. It was noted that a precursor adduct other than the molecular mass ion for a specific vitamin D analogue can produce a more abundant MS1 signal and that the ESI source parameters vary between analytes with different chemical properties and should therefore be optimised individually for each analyte. Various columns were assessed and sufficient chromatographic separation between the relevant analytes was achieved with an Agilent Technologies Pentafluorophenyl column. Baseline separation was achieved between 25(OH)D3 and 3-epi-25(OH)D3 as well as 25(OH)D2 and 3-epi-25(OH)D2, which is a requirement for this method to be viable. The method was subjected to a series of validation steps to ensure the accuracy and precision of the method. These included the assessment of the analytical range, LOD, LOQ, inaccuracy, imprecision, stability, interference and recovery. It was found that the optimised method had good linearity (r > 0.995), acceptable repeatability (CV < 10 %) and within-lab precision (CV < 15%) and excellent method accuracy (systematic error < 6.60 %). Furthermore, all the analytes proved to be stable for 48 hours after sample preparation with no interferences found for co-eluting analytes. Finally, based on the sigma metric scale specifications, it was calculated that this method proved to be “world class” and very little QC is needed to ensure the quality of the data derived from this method. Based on the findings in this study, it was concluded that a novel LC-MS/MS method for the quantification of twelve vitamin D analogues in a single run was successfully developed. All the LC-MS/MS parameters were optimised to ensure optimal analytical sensitivity for each analyte and the method was validated based on a series of method validation steps required for implementation into a clinical laboratory. This validation proved this method to be ready for implementation into a clinical environment. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015

In the early 19th century a ground-breaking discovery was made that linked a dietary deficiency of a fat-soluble vitamin with the childhood disease known as rickets. The vitamin was named vitamin D and extensive research regarding the physiological importance of this vitamin followed ever since. It is currently known that vitamin D plays an important role in maintaining the calcium and phosphate homeostasis in the human body. Less clear evidence states the medical importance of vitamin D in the prevention and cancer, autoimmune disease and diabetes. Current literature shows that vitamin D has five distinct forms, vitamin D1 to D5, of which vitamin D2 and D3 are the most studied forms. The term “vitamin D” is often wrongfully used to include the vitamin D mother molecule, the vitamin D status indicator (25(OH)D), the biologically active form (1,25(OH)2D) and biologically inactive form (24,25(OH)2D). The interest for measurement of these vitamin D analogues is a continuously growing field both on individual and epidemiological level. For decades laboratories have struggled to produce a robust method capable of quantifying these different vitamin D analogues and uncovered a new form of complexity regarding the analysis of these analogues. The identification of the C3-epimeric forms of vitamin D metabolites has forced laboratories to rethink their analytical methods and several concerns were raised regarding the overestimation of the true vitamin D status by current analytical methods. The quantification of the biologically active and inactive forms of vitamin D is reported to be difficult and to date very few LC-MS/MS methods reported in the literature are able to quantify various vitamin D analogues. However, to our knowledge none of these methods are able to include the precursor vitamin D, the 25-hydroxylated metabolites, the biologically active and inactive metabolites, C3-epimers and isobaric compounds in a single run. Therefore the aim of this study was to develop, optimise and validate a LC-MS/MS method for the quantification of twelve vitamin D analogues in a single run. This was done by optimising the underlying LC-MS/MS parameters to ensure optimal analytical sensitivity in positive ESI mode and sufficient chromatographic separation between analytes with similar chemical properties. Furthermore, the optimised method was validated to ensure the accuracy and precision of the method before implementation into a clinical environment. The vitamin D analogues included in this study were vitamin D2, vitamin D3, 25(OH)D2, 25(OH)D3, 1,25(OH)2D2, 1,25(OH)2D3, 24,25(OH)2D2, 24,25(OH)2D3, 3-epi-25(OH)D2, 3-epi- 25(OH)D3, 7(OH)4C3 and 1α(OH)D3. A double liquid-liquid extraction with hexane and ethyl acetate were found to be the most efficient at extracting the vitamin D analogues from a serum matrix after matrix modification with sodium hydroxide. Recoveries of > 95 % (CV <10 %) were achieved for all the analytes. It was noted that a precursor adduct other than the molecular mass ion for a specific vitamin D analogue can produce a more abundant MS1 signal and that the ESI source parameters vary between analytes with different chemical properties and should therefore be optimised individually for each analyte. Various columns were assessed and sufficient chromatographic separation between the relevant analytes was achieved with an Agilent Technologies Pentafluorophenyl column. Baseline separation was achieved between 25(OH)D3 and 3-epi-25(OH)D3 as well as 25(OH)D2 and 3-epi-25(OH)D2, which is a requirement for this method to be viable. The method was subjected to a series of validation steps to ensure the accuracy and precision of the method. These included the assessment of the analytical range, LOD, LOQ, inaccuracy, imprecision, stability, interference and recovery. It was found that the optimised method had good linearity (r > 0.995), acceptable repeatability (CV < 10 %) and within-lab precision (CV < 15%) and excellent method accuracy (systematic error < 6.60 %). Furthermore, all the analytes proved to be stable for 48 hours after sample preparation with no interferences found for co-eluting analytes. Finally, based on the sigma metric scale specifications, it was calculated that this method proved to be “world class” and very little QC is needed to ensure the quality of the data derived from this method. Based on the findings in this study, it was concluded that a novel LC-MS/MS method for the quantification of twelve vitamin D analogues in a single run was successfully developed. All the LC-MS/MS parameters were optimised to ensure optimal analytical sensitivity for each analyte and the method was validated based on a series of method validation steps required for implementation into a clinical laboratory. This validation proved this method to be ready for implementation into a clinical environment. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015

Multiple sclerosis is a complex immune mediated condition of the central nervous system characterized by myelin loss and progressive neurodegeneration. The risk of developing MS is influenced by both genetic and environmental agents and, among them, several lines of evidence support a role for vitamin D deficiency, Epstein-Barr virus (EBV) infection and smoking in the aetiology of this disease. The aim of this work was to further elucidate how nature and nurture act in the causal cascade leading to MS. In chapter 1, I show that the main genetic factor in adult MS (the HLA-DRB1*1501 allele) plays an equally important role in paediatric cases of MS (PMS) and that EBV negative PMS patients represent a separate entity characterized by lower age at disease onset, lower female to male ratio and a trend towards a lower frequency of the HLA-DRB1*1501 allele. In chapter 2, I provide evidence in support of month of birth having a role on MS risk and T cell production and that vitamin D may underlie this effect. In chapter 3 I demonstrate the presence of a link between vitamin D deficiency and the immune response against EBV, whereby the proportion of EBV seropositive MS patients and controls increases with increasing latitude and high dose vitamin D supplementation appears to reduce the level of antibodies against this virus. In chapter 4, I show that MS associated genetic variants are located in genomic regions that exert a regulatory function and are active in immune cell types. In chapter 5, I illustrate how vitamin D receptor binding is also located within active regulatory regions in immune cells and that this is particularly evident near MS associated genes. Finally, in chapter 6, I use chromatin data on more than 100 different cell types and conclude that MS associated genetic variants are particularly active in T helper, T cytotoxic and B cells. Further work is needed to elucidate how genetic and environmental agents play a role in the cause of MS and to develop effective strategies for disease treatment and prevention.

Vitamin D is an essential nutrient in the human diet. A unique property of vitamin D is that it can be produced by endogenous synthesis in the skin following sufficient Ultraviolet B (UVB) radiation. In fact, our understanding of this compound has changed, such that it is no longer consider a true vitamin, but rather a steroid hormone. De-identified data for this analysis were derived from women residing in Jeddah, Saudi Arabia who completed routine medical visits in the summer of 2009 at King Fahad Hospital (KFH). In Chapter 1,“THE ASSOCIATION BETWEEN VITAMIN D STATUS IN NORMAL WEIGHT VERSUS OBESE WOMEN RESIDING IN WESTERN SAUDI ARABIA” we evaluate the relationship between body size and serum 25(OH)D concentrations including the association between change in body size during adulthood and vitamin D status. This study examines whether the current weight and weight change since age 18 years are associated with vitamin D status. This study found that neither current weight nor adult weight gain were associated with vitamin D status in Saudi Arabian women. In chapter 2,“IS AVOIDING SUN EXPOSURE VIA SUN PROTECTION PRACTICES ASSOCIATED WITH LOW VITAMIN D STATUS IN SAUDI ARABIAN WOMEN?” we investigate whether women who avoid UV exposure have lower 25(OH)D concentrations than women who do not avoid exposure. UV exposure was defined by time in outdoor activities, use of protective clothing and sunscreen. This study demonstrated that avoiding UV exposure via indoor activity and the use of sunscreen or/and wearing protective clothing was not associated with vitamin D status. Chapter 3, “VITAMIN D STATUS AND BREAST CANCER IN SAUDI ARABIAN WOMEN: A CASE CONTROL STUDY” we examine if vitamin D status as assessed by serum concentrations of 25(OH)D would be lower in breast cancer cases as compared to controls. This study demonstrated that there is a significant relationship between higher serum concentrations of 25(OH)D and lower risk of breast cancer. Chapter 4, “IMPLICATIONS AND FUTURE DIRECTIONS” is presented a summary of key findings from the three studies in this dissertation to determine avenues of further research. The appendices consist of materials related to the dissertation work.

Background: Many studies have reported that adherence to health promotion guidelines for diet, physical activity, and maintenance of healthy body weight may decrease cancer incidence and mortality, including site-specific cancers such as colorectal cancer. To date, there have been no studies investigating adherence to the American Cancer Society's (ACS) Nutrition and Physical Activity Cancer Prevention Guidelines and the development and characteristics of premalignant lesions. Several individual lifestyle factors targeted by the ACS guidelines have also been associated with circulating concentrations of vitamin D metabolites. These associations suggest that adherence to the ACS guidelines may be related to improved vitamin D status. This dissertation sought to 1) synthesize the evidence from published prospective cohort studies regarding adherence to the ACS and World Cancer Research Fund/American Institute for Cancer Research (WCRF/AICR) nutrition and physical activity cancer prevention guidelines and the risk of overall cancer incidence and/or cancer mortality and 2) to further explore the role of adhering to a healthy lifestyle pattern as outlined by the ACS guidelines on a) colorectal adenoma occurrence and b) circulating concentrations of vitamin D metabolites using secondary data analyses from completed large prevention trials. Methods: A systematic review was performed to examine associations between adherence to established cancer prevention guidelines for diet and physical activity and overall cancer incidence and mortality. PubMed, Google Scholar, and Cochrane Reviews databases were searched following the current recommendations of Preferred Reporting Items for Systematic Reviews and Meta-analysis Approach (PRISMA). Cross-sectional and prospective analyses of pooled participants were also conducted from the Wheat Bran Fiber (n=503) and Ursodeoxycholic Acid (n=854) trials. A cumulative adherence score was constructed using baseline data regarding body size, diet, physical activity, and alcohol consumption. Continuous vitamin D metabolite concentrations and clinically significant vitamin D categories were evaluated with adherence score category using multiple linear and logistic regression models, respectively. Baseline adenoma characteristics and new colorectal adenomas were evaluated by adherence score category using multivariate logistic regression models. Results: Twelve studies met inclusion criteria for the systematic review. High versus low adherence to established nutrition and physical activity cancer prevention guidelines was consistently and significantly associated with decreases of 10-61% in overall cancer incidence and mortality. Consistent significant reductions were also shown for breast cancer incidence (19-60%), endometrial cancer incidence (23-60%), and colorectal cancer incidence in both men and women (27-52%). Findings for lung cancer incidence were equivocal and no significant relationships were found between adherence and ovarian or prostate cancer. In the pooled analyses, concentrations of circulating 25-hydroxycholecalciferol [25(OH)D] were statistically significantly higher among participants with high versus low adherence to guidelines (31.4 ± 0.8 and 26.3 ± 0.8 ng/ml, respectively; p<0.001). For 1,25(OH)₂D concentrations, high adherence was again significantly related to greater metabolite levels, with mean concentrations of 36.4 ± 1.1 and 31.2 ± 1.2 pg/mL for high- and low-adherers, respectively (p<0.001). Furthermore, the odds of attaining sufficient 25(OH)D status were 4.30 times higher for those most adherent versus those least adherent (95% CI: 2.43-7.60). Significantly reduced odds of having three or more adenomas at baseline were shown for moderate (odds ratio [OR]=0.67, 95% confidence intervals [CI]: 0.46-0.99) and highly adherent (OR=0.50, 95% CI: 0.31-0.81) participants compared to those with low adherence (p-trend=0.005). Conversely, guideline adherence was not associated with the development of a new colorectal adenoma (moderate adherence OR=1.16, 95% CI: 0.85-1.59, high adherence OR=1.23, 95% CI: 0.85-1.79). Conclusion: From the systematic review, greater adherence to cancer prevention guidelines for diet and physical activity was consistently associated with lower risks of overall cancer incidence and mortality, including for some site-specific cancers. In addition, adherence to the ACS guidelines was associated with higher concentrations of both of 25(OH)D and 1,25(OH)₂D. Following the ACS guidelines could potentially increase 25(OH)D levels as much as that observed by a supplement of 1000 IU/d in a population similar to ours, and therefore may be a viable strategy for increasing both 25(OH)D and 1,25(OH)₂D concentrations. Further, our findings suggest that following the ACS Nutrition and Physical Activity guidelines may lead to a lower odds of multiple adenomas when at least one adenoma is detected. Finally, these guidelines and recommendations are consistent with strategies for the prevention of major diseases, and if followed, will ultimately lead to healthier lives overall.

Background: Previous research suggests that vitamin D deficiency during pregnancy may be associated with suboptimal fetal growth, but direct evidence is lacking. Our objectives were 1) to develop a method for measurement of the fetal sphenoidal fontanelle area (FA) and femur volume (FV) using 3D ultrasound; 2) to create normal charts for FA and FV; and 3) to correlate FA and FV with maternal vitamin D concentration. Methods: FA measurement in 3D was evaluated in vitro and in vivo. Different segmentation methods for FV measurement were explored. A novel FV method was described which consists of three linear measurements and a volume equation; this was validated in vitro and also by comparing FV measured sonographically to the true volume assessed by computed tomography (CT), in 6 cases following pregnancy termination. A cohort of 868 uncomplicated pregnancies was selected on the basis of strict inclusion criteria; participants underwent serial ultrasound scans for FV and multilevel modeling was used for the creation of a “prescriptive” FV chart. Finally, a different cohort of 357 healthy pregnant women had serum vitamin D levels and FV ultrasound at 34 weeks gestation and dual emission x-ray absorptiometry (DEXA) of their neonates in order to investigate the prenatal determinants of fetal bone mass. Results: FA measurement was accurate in vitro, but unreliable in vivo and was therefore abandoned. A novel FV method had excellent agreement with CT and superior repeatability compared with segmentation-based methods. A normal FV chart was created and the regression equations for the median and percentile values were presented. Vitamin D demonstrated a significant correlation with FV. Conclusions: FV is a reliable sonographic marker of skeletal growth. Maternal vitamin D deficiency is associated with reduced FV. This finding has public health implications as reduced bone mass may increase the lifetime risk of osteoporosis, through fetal programming.