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Efficacy and safety of two doses of vitamin D supplementation (400 or 2000 IU/D) in pediatric Crohn's… Wingate, Kirstin Emma 2013

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EFFICACY AND SAFETY OF TWO DOSES OF VITAMIN D SUPPLEMENTATION (400 OR 2000 IU/D) IN PEDIATRIC CROHN’S DISEASE: A RANDOMIZED CONTROLLED TRIAL  by Kirstin Emma Wingate  B.Sc. in F.N.H., University of British Columbia, 2010  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Human Nutrition)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  April 2013  © Kirstin Emma Wingate, 2013  Abstract BACKGROUND: Vitamin D is important for children with Crohn’s disease for the promotion of optimal bone health and because suboptimal vitamin D has been associated with increased disease activity through epidemiological and animal studies. Health Canada’s vitamin D Recommended Dietary Allowance for healthy children is 600 IU/d to promote serum 25-hydroxyvitamin D (25OHD) concentrations >50 nmol/L. There is little experimental evidence informing the dose of vitamin D required for children with Crohn’s disease. OBJECTIVE: We aimed to determine whether a vitamin D3 supplement of 2000 IU/day would achieve a higher prevalence of serum 25OHD concentrations >50 or 75 nmol/L compared to 400 IU/day, in children with quiescent Crohn’s disease. Exploratory objectives included assessing any difference between groups in the bone formation biomarker, bone specific alkaline phosphatase (BSAP), concentrations and disease activity, as measured by the Pediatric Crohn’s Disease Activity Index (PCDAI). METHODS: Eighty-three children with quiescent Crohn’s disease (PCDAI <10) were recruited from British Columbia Children’s Hospital and McMaster Children’s Hospital and randomized to receive 400 or 2000 IU/d vitamin D3 for 6 months. Clinical and biochemical data were collected at baseline, 3 months and 6 months. RESULTS: Using an intention to treat analysis, at 6 months subjects receiving 2000 IU (vs. 400 IU) were more likely to achieve serum 25OHD concentrations >75 nmol/L (79% vs. 35%, P<0.001), but proportions >50 nmol/L did not differ (88% vs. 87%, p = 0.934). After adjustment for baseline 25OHD, mean serum 25OHD was higher in the 2000 IU group than the 400 IU group by 24 (95% CI: 15 – 33) nmol/L. Regimens were found to be equally safe  ii  based on the assessment of serum calcium and phosphate concentrations and urinary calcium to creatinine ratio, and no effect of vitamin D supplementation was observed on secondary objective outcome measures of serum BSAP concentration, or the PCDAI score. CONCLUSION: A vitamin D3 supplement dose of 2000 IU/d is more effective than 400 IU/d in promoting serum 25OHD concentrations >75nmol/L in children with quiescent Crohn’s disease, however both regimens are effective and safe in promoting concentrations greater than the current cutoff for adequacy of 50 nmol/L.  iii  Preface This thesis presents work conducted by myself, Kirstin Wingate, under the supervision of Dr. Tim Green, with ongoing guidance from Dr. Kevan Jacobson and Dr. Yvonne Lamers at the University of British Columbia. This project required significant collaboration and effort from medical and research personnel in both Vancouver and Hamilton. Study protocol was designed by principal investigator Dr. Tim Green and coinvestigator Dr. Kevan Jacobson in Vancouver. Dr. Robert Issenman was the responsible physician and principal investigator at the McMaster Children’s Hospital site. Additional contributions included the use and analysis of the validated food frequency questionnaire from Dr. Susan Whiting at the University of Saskatchewan, and conduct of serum 25OHD and BSAP analyses by the laboratory of Dr. Hope Weiler at McGill University. My role in this project included the overall coordination and execution of the study protocol between sites. I was the primary individual responsible for recruitment, enrolment and data collection in Vancouver. Ultimately I conducted sample processing and data analysis for all 83 subjects, and finally the writing of this thesis. The content of this thesis will be condensed and submitted to academic journals for publication. The University of British Columbia Children and Women’s Clinical Research Ethics Board and the Hamilton Health Sciences/Faculty of Health Sciences Research Ethics Board approved this study protocol (identifiers: H10-01391 and 10-393 respectively). This study is also registered at ClinicalTrials.gov (identifier: NCT01187459). The University of British Columbia Vitamin Research Fund financed this study.  iv  Table of Contents Abstract.................................................................................................................................... ii	
   Preface ..................................................................................................................................... iv	
   Table of Contents .................................................................................................................... v	
   List of Tables ......................................................................................................................... vii	
   List of Figures....................................................................................................................... viii	
   List of Abbreviations ............................................................................................................. ix	
   Acknowledgements ................................................................................................................. x	
   Chapter 1: Literature Review ............................................................................................... 1	
   1.1	
   Crohn’s Disease ........................................................................................................... 1	
   1.1.1	
   Epidemiology of Crohn’s Disease ........................................................................ 1	
   1.1.2	
   Symptoms and Impact of Crohn’s Disease ........................................................... 2	
   1.1.3	
   Medical Management of Crohn’s Disease ............................................................ 3	
   1.2	
   Vitamin D Overview .................................................................................................... 4	
   1.2.1	
   Vitamin D Endogenous Synthesis ........................................................................ 5	
   1.2.2	
   Food Sources and Absorption of Vitamin D......................................................... 5	
   1.2.3	
   Activation and Transport of Vitamin D ................................................................ 7	
   1.2.4	
   Vitamin D Action in the Body .............................................................................. 9	
   1.2.4.1	
   Bone Metabolism Overview .......................................................................... 9	
   1.2.4.2	
   Vitamin D in Calcium, Phosphorus and Bone Metabolism ........................... 9	
   1.2.4.3	
   Non-Classical Vitamin D Functions ............................................................ 10	
   1.2.5	
   Vitamin D Dietary Reference Intake .................................................................. 12	
   1.2.6	
   Assessment of Vitamin D Status......................................................................... 13	
   1.2.6.1	
   Serum 25OHD Cutoff for Vitamin D Status Assessment............................ 14	
   1.2.6.2	
   Determinants of Vitamin D Status ............................................................... 15	
   1.3	
   Vitamin D Status of Children with Crohn’s Disease ................................................. 15	
   1.4	
   Vitamin D and Bone Health in Crohn’s Disease ....................................................... 19	
   1.4.1	
   Bone Specific Alkaline Phosphatase in Children with Crohn’s Disease ............ 20	
   1.5	
   The Role of Vitamin D in Crohn’s Disease ............................................................... 21	
   1.5.1	
   Epidemiologic Observations ............................................................................... 21	
   1.5.2	
   Basic Research .................................................................................................... 22	
   1.5.3	
   Prospective Human Research ............................................................................. 24	
   1.6	
   Summary of Evidence and Gaps to be Addressed in this Thesis ............................... 26	
   1.7	
   Research Objectives ................................................................................................... 28	
   Chapter 2: Research Methods ............................................................................................ 29	
   2.1	
   Purpose....................................................................................................................... 29	
   2.2	
   Overview .................................................................................................................... 29	
   2.3	
   Sample Size................................................................................................................ 29	
   2.4	
   Participant Selection and Recruitment ....................................................................... 29	
   2.5	
   Procedures .................................................................................................................. 31	
   2.6	
   Dietary Supplements, Randomization, Adherence .................................................... 32	
   2.7	
   Dietary Assessment.................................................................................................... 34	
   2.8	
   Biochemical Measures ............................................................................................... 35	
   2.8.1	
   Collection and Processing of Blood and Urine Samples .................................... 35	
   2.8.2	
   Analysis of Serum and Urine Samples ............................................................... 35	
   v  2.9	
   Data Analysis ............................................................................................................. 38	
   Chapter 3: Results................................................................................................................ 40	
   3.1	
   Recruitment and Randomization................................................................................ 40	
   3.2	
   Adherence .................................................................................................................. 41	
   3.3	
   Baseline Characteristics ............................................................................................. 42	
   3.3.1	
   Dietary Intake...................................................................................................... 43	
   3.3.2	
   Baseline 25OHD by Participant Characteristic................................................... 44	
   3.4	
   Proportion of Subjects Achieving 25OHD Cutoffs ................................................... 45	
   3.5	
   Mean Serum 25OHD Concentrations ........................................................................ 48	
   3.5.1	
   Unadjusted Serum 25OHD Concentrations ........................................................ 48	
   3.5.2	
   Serum 25OHD Concentrations Adjusted for Baseline Values ........................... 49	
   3.6	
   BSAP and Disease Activity Results .......................................................................... 51	
   3.6.1	
   Adjusted Mean Serum BSAP ............................................................................. 51	
   3.6.2	
   Disease activity ................................................................................................... 53	
   3.7	
   Safety Markers ........................................................................................................... 53	
   Chapter 4: Discussion .......................................................................................................... 58	
   4.1	
   Subjects achieving serum 25OHD concentration cutoffs .......................................... 58	
   4.2	
   Difference in Mean 25OHD Concentrations between Groups .................................. 59	
   4.3	
   Baseline 25OHD Concentrations ............................................................................... 60	
   4.4	
   Dietary Assessment.................................................................................................... 62	
   4.5	
   Safety of Treatment Regimens in this Population ..................................................... 62	
   4.6	
   PCDAI ....................................................................................................................... 63	
   4.7	
   BSAP ......................................................................................................................... 63	
   4.8	
   Limitations ................................................................................................................. 64	
   4.9	
   Directions for Future Research .................................................................................. 65	
   Chapter 5: Conclusion ......................................................................................................... 68	
   References .............................................................................................................................. 70	
   Appendices ............................................................................................................................. 79	
   Appendix A Study Documents ........................................................................................... 79	
   A.1	
   Letter of First Contact ........................................................................................... 79	
   A.2	
   Study Consent Form.............................................................................................. 80	
   A.3	
   Study Assent Form ................................................................................................ 86	
   Appendix B Pediatric Crohn’s Disease Activity Index ...................................................... 90	
   Appendix C Food Frequency Questionnaire....................................................................... 92	
    vi  List of Tables Table 1.1. Vitamin D Recommended Dietary Allowance and Tolerable Upper Intake per age group ……………………………………………………………………………….13 Table 1.2. Reported Vitamin D status of children with inflammatory bowel disease …17 Table 2.1. Ingredient list of the study multivitamin ………………………………………33 Table 2.2. Vitamin D supplement dosage quality control (IU) ………………………33 Table 2.3. Summary of 25OHD intra-assay coefficient of variation (CV%) ……………36 Table 2.4. Normal ranges for biochemical measures ……………………………………38 Table 3.1 Adherence as reported by subjects, N (%) ……………………………………42 Table 3.2 Subject baseline characteristics …………………………………………………..43 Table 3.3. Serum 25OHD concentration, by predictor ……………………………………..44 Table 3.4 Proportion of subjects achieving 25OHD cutoffs, ITT …………………………46 Table 3.5 Proportion of subjects achieving 25OHD cutoffs, as treated …………………….46 Table 3.6 Proportion of subjects achieving 25OHD cutoffs, 80% adherence ……………47 Table 3.7 Mean outcome measure per group, ITT ………………………………………….49 Table 3.8 Mean outcome measure per group, as-treated ……………………………………49 Table 3.9 Difference in outcome measures adjusted for baseline values, ITT ……………..50 Table 3.10 Difference in outcome measures adjusted for baseline values, as treated ……50 Table 3.11 Disease activity by group at each study visit, ITT …………………………52 Table 3.12 Disease activity by group at each study visit, as treated ……………………..52 Table 3.13 Number of subjects exceeding safety marker cutoffs, ITT ……………………..54 Table 3.14 Number of subjects exceeding safety marker cutoffs, as-treated ……………….55 Table 3.15. Mean serum and urine laboratory analyses at each study visit, ITT …………56 Table 3.16. Mean serum and urine laboratory analyses at each study visit, as treated ……..57  vii  List of Figures Figure 1.1 Chemical structure of vitamin D …………………………………………………4 Figure 1.2 Chemical structure of vitamin D2 ………………………………………………...6 Figure 1.3 Chemical structure of 25-hydroxyvitamin D3 …………………………………….8 Figure 1.4 Chemical structure of 1,25-dihydroxyvitamin D3 ………………………………...8 Figure 3.1 Subject flow diagram …………………………………………………………....41  viii  List of Abbreviations 1,25(OH)2D 25OHD ANOVA BC BSAP CI CDAI CV DBP DSS ESR FFQ IBD IL-10 IOM ITT NOD2 PCDAI PTH RDA SD UL UV VDR WT  1α,25-dihydroxyvitamin D 25-hydroxyvitamin D Analysis of variance British Columbia Bone specific alkaline phosphatase Confidence interval Crohn’s Disease Activity Index Coefficient of variation Vitamin D binding protein Dextran sodium sulfate Erythrocyte sedimentation rate Food frequency questionnaire Inflammatory bowel disease Interleukin 10 Institute of Medicine Intent to treat Nucleotide-binding oligomerization domain-containing protein 2 Pediatric Crohn’s Disease Activity Index Parathyroid hormone Recommended Dietary Allowance Standard deviation Tolerable Upper Intake Level Ultraviolet Vitamin D receptor Wildtype  ix  Acknowledgements First and foremost I would like to thank my supervisor Dr. Tim Green for his ongoing guidance and mentorship. He is largely responsible for the positive learning experience I have had throughout this process. I am grateful to Dr. Yvonne Lamers for the expertise, perspective, and attention to detail she brought to the project, and to Dr. Jacobson for contributing his guidance, medical knowledge and supporting me with his resources at BC Children’s Hospital. I would like to acknowledge the source of my stipend, the University of British Columbia Vitamin Research Fund, without which I would not have been able undertake this program. I would like to recognize the study participants whose commitment to this project was above and beyond my expectations. Many medical, research and administrative staff at both study sites contributed their time and energy to help me with this project, without which it would not have been possible. I thank Dr. Robert Issenman, and his research assistants Rachel Vizcarra, Jessica Blavignac and Jenny Yip at the Hamilton site for their excellent data collection. In Vancouver I am grateful to Tina Li, Nicole Curtis, Samira Dadgar and Debbie van den Brink for helping me when the study workload became too heavy. John Bhullar, Josephine Baldwin and Sherry Agellon have been my contacts at their respective laboratories, and have each been exceedingly helpful. The moral support of my lab mates Kaitlin March and Nancy Chen has been invaluable, and has enriched my time in this program. Finally, to my loving and encouraging parents, I will forever appreciate the financial, moral and emotional support you have given me throughout my undergraduate and now graduate training.  x  Chapter 1: Literature Review 1.1  Crohn’s Disease Inflammatory bowel disease (IBD) refers to a group of diseases in which the  gastrointestinal tract suffers lesions and ulcerations as a result of chronic inflammation. Crohn’s disease distinguishes itself from other IBD such as ulcerative colitis in that it may affect any section of the intestine, although it is most commonly seen in the ileum and colon (1). In Crohn’s disease, unlike ulcerative colitis, areas of inflammation may be discontinuous and penetrate deep into the epithelial layers of the intestine, even leading to development of fistulas (1). Impact of the disease can be wide ranging and can severely impair a patient’s overall health and quality of life. The etiology of Crohn’s disease is an area of ongoing research. Crohn’s disease is thought to occur in genetically predisposed individuals who suffer from an inappropriate immune response to commensal bacteria in the gastrointestinal tract (2). Current genome-wide association studies have identified over 99 loci of genetic risk for IBD and 71 for Crohn’s disease (2). Twin studies have shown a concordance of only 30-35% for Crohn’s disease in identical twins, suggesting that non-genetic factors also play a significant role in disease development (2,3). Smoking is one environmental factor identified as a disease modifier by increasing risk in those genetically predisposed to the development of Crohn’s disease (4,5). 1.1.1  Epidemiology of Crohn’s Disease Incidence and prevalence data on Crohn’s disease varies widely. A comprehensive  systematic review of 262 population-based prevalence and incidence IBD studies published worldwide reported that North American prevalence ranged from 16.7 to 318.5 per 100,000 (6). A similar prevalence was estimated for Europe, ranging from 0.6 to 322 per 100,000, while for  1  Asia estimates ranged from 0.88 to only 67.9 per 100,000 (6). These particularly wide ranges represent the diversity of studies included in this review, occasionally reporting incidence and prevalence in small geographic areas. The authors of this review reported increasing Crohn’s disease incidence rates over time in 75% of studies (6). Geographic distribution of this trend could potentially indicate important environmental factors in disease development, such as a positive association between urbanization and Crohn’s disease. However this trend is unfortunately overshadowed by the confounding factor of increased IBD awareness and improved diagnostic measures in healthcare systems globally. Canada has one of the highest reported prevalence rates of Crohn’s disease worldwide (6). Estimates from health databases from five Canadian provinces reported rates from 161 per 100,000 in British Columbia to 319 per 100,000 in Nova Scotia (7). Given that 33.4 million people live in Canada, this translates into 50,000 to 100,000 cases nation-wide (8). Childhood or adolescent onset of Crohn’s disease occurs in up to 20% of patients (9,10). The pediatric prevalence of Crohn’s disease in Canada is estimated to range from 30.5 to 71.1 per 100,000, based on health database information from 2000 (7). 1.1.2  Symptoms and Impact of Crohn’s Disease Symptoms of individuals with Crohn’s disease can vary from mild to severe and include  diarrhea with or without blood, abdominal pain, fatigue, anorexia, and weight loss or poor weight gain (11). Extraintestinal manifestations occur quite frequently and include fever, mouth ulcers, arthritis, skin rash (erythema nodosum or pyoderma gangrenousum), kidney stones, hepatobiliary disease (autoimmune hepatitis or primary sclerosis cholangitis) or ophthalmologic involvement (11,12). Individuals suffering from Crohn’s disease may experience nutritional deficiencies, a consequence most serious in the pediatric population (11).  Growth failure, subsequent to  inflammation and suboptimal nutrient absorption, is reported to occur in 10 to 56% of pediatric 2  Crohn’s disease cases (13). Consequences can include delayed puberty, which can slow normal bone mineral accumulation contributing to osteoporosis, diminish final adult height, as well as have psychological and self-esteem consequences for the adolescent (14–16). While many patients are able to attain clinical remission through medical and/or surgical therapies, there remains a high probability of relapse or flare-up due to the chronic and recurring nature of the disease (17). 1.1.3  Medical Management of Crohn’s Disease Phenotype, treatment and disease course for Crohn’s disease are largely similar between  adults and children, although greater colonic involvement is often seen in children under 10 years of age than in older cases (18,19). Most Crohn’s disease follows a chronic, relapsing disease course, while approximately 13% of patients may have an unremitting disease course, and 10% may achieve prolonged remission (17). Goals of medical therapy of pediatric Crohn’s disease include maintaining clinical remission, achieving adequate growth, minimizing adverse effects, and promoting intestinal healing (19). Course of treatment depends on the severity of disease. Corticosteroids are used in the short-term for moderate to severe symptom relief, however longterm treatment with corticosteroids is generally considered inappropriate due to side effects which can include impaired growth and reduced bone mineral density (11,19,20). Immunomodulators, biologic therapies, and 5-aminosalicylate drugs can be used to maintain remission in the longer term (11,19). Surgical resection is performed in up to 80% of adult cases to remove areas of severe disease involvement; however, the disease will reoccur at or near the site of surgery in approximately 30% of patients after 10 years (21,22). Surgery is also used as a course of treatment for refractory pediatric cases (18,19). Disease activity, a measure of severity, is generally assessed by the Crohn’s Disease Activity Index in adults, or the Pediatric Crohn’s Disease Activity Index (PCDAI) in children. 3  The PCDAI was developed by a group of pediatric IBD experts at a consensus meeting in 1990 and validated at 12 North American institutions (9,23). The index can be used to monitor a patient’s response to a medical regimen over time, as well as to compare severity of disease between patients (23). The index will be described in further detail in the methods section of this thesis.  1.2  Vitamin D Overview Vitamin D is a prohormone essential for calcium metabolism and bone mineralization,  with an emerging role in immune system functioning. Deficiency is known to cause rickets in children and osteomalacia in adults. It is a secosteroid, in that is has a chemical structure containing four cycloalkane rings, one of which is broken (Figure 1.1). Vitamin D can be obtained through dietary sources or produced endogenously when the skin is exposed to ultraviolet light.  Seco-ring B  OH  Figure 1.1. Chemical structure of vitamin D3 (24,25).  4  1.2.1  Vitamin D Endogenous Synthesis The steroid 7-dehydrocholesterol is synthesized in the sebaceous glands of the skin.  Double bonds in ring B of the compound are able to absorb photons during exposure to ultraviolet B light, of wavelength 290 to 315 nm (26). This event causes ring B to open, forming precholecalciferol, or previtamin D3. Through thermal isomerization, the double bonds on previtamin D are rearranged over the following few days, producing cholecalciferol, or vitamin D3 (Figure 1.1) (26). Cholecalciferol diffuses into the bloodstream, where it is transported by the vitamin D binding protein. 1.2.2  Food Sources and Absorption of Vitamin D Vitamin D can also be obtained from dietary sources, primarily sources of animal origin.  The highest naturally occuring sources of cholecalciferol, or vitamin D3, are fatty fish such as salmon, tuna and sardines (24). Fungal sources of vitamin D such as mushrooms or yeast contain ergocalciferol, or vitamin D2 (Figure 1.2), synthesized from ergosterol upon ultraviolet irradiation (24). Vitamin D2 and vitamin D3, together designated vitamin D, are both effective in preventing deficiency diseases such as rickets, however vitamin D3 is generally considered preferable due to its greater bioefficacy (27). In Canada and the United States, milk and milk alternatives are fortified to a target level of 41 IU vitamin D/100 mL of primarily vitamin D3 (28,29). Other food products that may be fortified with vitamin D include orange juice, margarine, yogurt, cheese, breads and cereals (24).  5  OH  Figure 1.2. Chemical structure of vitamin D2 (24,25).  Vitamin D is a fat-soluble vitamin, and is absorbed from the intestinal lumen as part of a micelle with the aid of bile salts (30). This absorption is assumed to occur by passive diffusion, however there is recent evidence that vitamin D may also make use of cholesterol transport proteins (31). Vitamin D is most efficiently absorbed when consumed with fats or large meals (32,33). Vitamin D absorption may be compromised in patients with IBD who may have decreased healthy intestinal surface area due to disease activity or surgical resection. Decreased absorption of vitamin D has been reported in Crohn’s disease patients post-intestinal resection (34). Vitamin D absorption has been found to be approximately 30% lower in subjects with Crohn’s disease than in a healthy control group, when serum vitamin D concentrations were measured 12 hours after administration of 50,000 IU of vitamin D2 (35). There were no significant associations between disease location, receiving surgery or not, or type of surgery, and ability to absorb vitamin D (35).  6  1.2.3  Activation and Transport of Vitamin D Once absorbed into the intestinal wall, the majority of dietary vitamin D is incorporated  into chylomicrons and enters the lymphatic circulatory system, eventually being delivered to the liver with the chylomicron remnants (24,36). Cholecalciferol that has been produced in the skin is bound to vitamin D binding protein (DBP) and transported in the bloodstream to the liver and other tissues such as adipose or muscle (36). Cholecalciferol and ergocalciferol are not biologically active. The first step in the activation of vitamin D occurs in the liver (37), where carbon 25 is hydroxylated by a hepatic cytochrome P-450, 25-hydroxylase, to form 25-hydroxyvitamin D (25OHD) (Figure 1.3) (38). This hydroxylation of vitamin D is poorly regulated; therefore levels of circulating 25OHD will increase as intake and endogenous synthesis of vitamin D increase (38). For this reason, measurement of plasma or serum 25OHD levels can be used to assess vitamin D status (26). The second step in activation of vitamin D, hydroxylation of 25OHD to form 1α,25-dihydroxyvitamin D (1,25(OH)2D) (Figure 1.4), is tightly regulated and occurs predominantly in the kidneys (39). The enzyme, 1α-hydroxylase, has been shown to be stimulated by parathyroid hormone action, and suppressed by increased dietary phosphate intake or elevated blood calcium or 1,25(OH)2D concentrations (38). 1,25(OH)2D is the biologically active form of vitamin D which carries out the many vitamin D actions in the body.  7  OH  OH  Figure 1.3. Chemical structure of 25-hydroxyvitamin D3 (25OHD3) (25,40).  OH  OH  OH  Figure 1.4. Chemical structure of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) (25,40).  As mentioned previously, vitamin D and its metabolites are fat soluble, and therefore require binding to transport proteins in order to be soluble in the bloodstream. DBP is the primary vitamin D transport protein, capable of binding vitamin D and its metabolites, in descending order of affinity: 25OHD, 24,25-dihydroxyvitamin D (24,25(OH)2D), 1,25(OH)2D, vitamin D (38). When bound to DBP, vitamin D metabolites are unable to enter tissue cells, and are in effect a stored reserve of vitamin D in the body (38,41). DBP plasma concentrations far  8  exceed vitamin D metabolite concentrations, and so >99% of circulating vitamin D metabolites are protein bound, essentially guarding against vitamin D intoxication (41,42). 1.2.4  Vitamin D Action in the Body  1.2.4.1  Bone Metabolism Overview Throughout the lifespan, bone is constantly being remodeled (43).  Bone resorption  occurs from the action of osteoclast cells, which act to dissolve bone matrix into its components (44). This serves to maintain skeletal mass, repair damage to bone, and maintain calcium homeostasis (45). Bone formation occurs from the action of osteoblast cells, which produce bone matrix proteins including type I collagen, as well as act to mineralize the bone matrix (44). In order to maintain bone mineral density in adults, the processes of bone formation and resorption need to be balanced (43,46). Disturbance to this balance can be caused by estrogen deficiency (as seen in post-menopausal women), normal metabolism changes seen in aging, corticosteroid use or by calcium or vitamin D deficiency and can lead to skeletal disorders such as osteoporosis (47). During childhood, a time of growth, bone undergoes modeling in addition to remodeling, wherein deposition of bone matrix exceeds its resorption (46). 1.2.4.2  Vitamin D in Calcium, Phosphorus and Bone Metabolism As the key mineral components of bone, adequate calcium and phosphorus are required  for bone formation (48,49). Serum calcium concentrations are tightly regulated in the body, to enable not only bone formation, but also muscle contraction, cell signaling, nerve transmission, and vasodilation and contraction (32). When low serum calcium concentrations are detected by receptors on the parathyroid gland and in the kidney, parathyroid hormone (PTH) secretion is increased (48). PTH promotes the conversion of 25OHD to 1,25(OH)2D in the kidney (48), and together these hormones act to increase low serum calcium concentrations through the following actions. 9  1,25(OH)2D exerts its functions by binding with the vitamin D receptor leading to the regulation of specific gene expression (48). At the intestinal level, 1,25(OH)2D interacts with the vitamin D receptor to increase calcium and phosphorus absorption (48). Calcium absorption increases from approximately 10-15% in the absence of vitamin D to 30-40% in its presence, while phosphorus absorption increases from 60% to approximately 80% (24). 1,25(OH)2D and the PTH stimulates osteoclast activity, leading to bone resorption and a release of calcium from the bone matrix into the bloodstream (48). Lastly, 1,25(OH)2D and PTH increase calcium reabsorption in renal tubules, reducing urinary calcium excretion and raising serum calcium concentrations (48). In vitamin D deficiency, there is disruption to the above metabolic pathways. Lack of vitamin D can lead to insufficient calcium-phosphorus product, impeding mineralization of bone (24). In adults, this manifests as osteomalacia, in children, as vitamin D deficiency rickets (24). The demineralized collagen matrix is prone to hydration and swelling, which can be painful, and in children, swollen joints and bone deformations can occur over time (50). Vitamin D deficiency has also been implicated in other skeletal complications including osteopenia, osteoporosis and risk of fracture (24). 1,25(OH)2D deficiency also causes parathyroid gland proliferation and PTH secretion, often leading to hyperparathyroidism in chronic kidney disease patients, which can be suppressed through 1,25(OH)2D therapy (38). 1.2.4.3  Non-Classical Vitamin D Functions The vitamin D receptor has been identified in many cell types including enterocytes,  osteoblasts, distal renal tubule cells, parathyroid gland cells, skin keratinocytes, promyelocytes, lymphocytes, colon cells, pituitary gland cells, and ovarian cells (48,51). In addition, many cell types have been found to convert 25OHD to 1,25(OH)2D, including antigen presenting cells, parasympathetic ganglia, hair follicles, the cerebral cortex and pancreatic islet cells (52,53). In 10  these cells, vitamin D has been shown to have diverse actions unrelated to calcium homeostasis, most which remain to be fully elucidated. Vitamin D deficiency has been related to breast, prostrate and colon cancer, and adequate vitamin D status has been found to prevent unregulated cell proliferation as seen in cancer (50). Increased risk of hypertension and cardiovascular disease has also been associated with vitamin D deficiency, although the effect of supplementation on these conditions remains uncertain (54,55). Vitamin D deficiency has been linked to autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus and multiple sclerosis (56,57). Vitamin D supplementation may even reduce risk of developing type 1 diabetes mellitus (58). 1,25(OH)2D has been shown to modulate both the innate and adaptive immune response through its targeting of immune cells including monocytes, macrophages, dendritic cells, T-lymphocyte and B-lymphocytes (59). In Crohn’s disease both the innate and adaptive immune responses are disregulated. Increased numbers of activated macrophages and dendritic cells are present in the lamina propria, and in turn there is increased production and circulation of proinflammatory cytokines and chemokines, particularly those that are stimulated by the NFκB, Type 1 T-helper cells (Th1) and Type 17 T-helper cell pathways (Th17) (60). There are also elevated levels of T-cell response cytokines such as IFN-γ, IL-17 and IL-21 (60). Among its many immunomodulatory properties, 1,25(OH)2D3 has been shown to inhibit macrophage and dendritic cell production of IL-12, a cytokine crucial for the development of Th1 cells, and furthermore to inhibit Th1 and Th17 production of IFN-γ and IL-17 (61–63). This overlap of 1,25(OH)2D3 action and the pathways involved in Crohn’s disease provides a foundation for a potential mechanistic link between vitamin D and Crohn’s disease.  11  1.2.5  Vitamin D Dietary Reference Intake In 2010, the Institute of Medicine (IOM), in partnership with Health Canada, conducted  an extensive review of the scientific literature relating to vitamin D, in order to update policy surrounding its recommended intake and assessment of status in individuals (41). New Recommended Daily Intakes were published based on promotion of optimal bone health. The authors of the report concluded that there was insufficient evidence to base intake recommendations for promotion of health outcomes other than bone health. The Recommended Dietary Allowance (RDA) is the recommended daily nutrient intake for the healthy population such that approximately 97.5% of individuals will meet their needs (32). For children and adults aged 9 to 70 years, the vitamin D RDA is 600 IU and the tolerable upper intake level (UL), the highest daily intake likely to pose no adverse health risks, is 4000 IU (Table 1.1) (32,64). Very little evidence exists to describe vitamin D intoxication in children. The current UL is an extrapolation of already scarce adult intoxication data (32). The UL set by Health Canada and the IOM in the United States is presented per age group in Table 1.1. The Estimated Average Requirement, the intake required such that 50% of the population will achieve the average vitamin D requirement, is 400 IU/d for children and youth.  12  Table 1.1 Vitamin D Recommended Dietary Allowance and Tolerable Upper Intake Level per age group Recommended Dietary Tolerable Upper Intake Age Group Allowance per day Level per day Infants 0-6 months  400 IU (10mcg)*  1000 IU (25 mcg)  Infants 7-12 months  400 IU (10mcg)*  1500 IU (38 mcg)  Children 1-3 years  600 IU (15 mcg)  2500 IU (63 mcg)  Children 4-8 years  600 IU (15 mcg)  3000 IU (75 mcg)  Children and Adults 9-70 years  600 IU (15 mcg)  4000 IU (100 mcg)  Adults > 70 years  800 IU (20 mcg)  4000 IU (100 mcg)  Pregnancy and Lactation  600 IU (15 mcg)  4000 IU (100 mcg)  *Adequate Intake rather than RDA 1.2.6  Assessment of Vitamin D Status As explained previously, 25OHD is the best indicator of vitamin D status from both  endogenous and exogenous sources. This marker is consistently used clinically and within the scientific research community (65). The active form of vitamin D, 1,25(OH)2D, is a poor measure of vitamin D status because its concentrations are tightly regulated, and can be increased during vitamin D deficiency when increased PTH upregulates vitamin D activation in the kidneys (65). There are different assays available for measuring 25OHD concentrations in serum, including competitive protein-binding assays, immunochemical assays, and chromatographic procedures, such as gas chromatography/ mass spectrometry (65). In this project a competitive chemiluminescence immunoassay was used to assess serum 25OHD concentrations, as described in further detail in the methods section.  13  1.2.6.1  Serum 25OHD Cutoff for Vitamin D Status Assessment Which serum 25OHD cutoff to use in determining adequate vitamin D status of  individuals is the subject of controversy. The cutoff for vitamin D deficiency is the level below which risk for rickets in children or osteomalacia in adults is increased (24). Children with vitamin D deficiency rickets will usually have 25OHD concentrations less than 10 nmol/L, and are considered to be deficient under 25 nmol/L (66). Other cutoffs, representing vitamin D insufficiency, have been used to describe a potential increase in risk for other disease outcomes. Various markers have been used to indicate potential vitamin D deficiency or insufficiency, including biochemical indices such as calcium absorption, PTH suppression or bone turnover markers as well as bone effects including bone mineral density and fracture risk (67). The interpretation of which outcomes are clinically relevant, and which cutoffs should be generalized to the population, is subjective. The IOM and Health Canada currently define deficiency as serum 25OHD concentrations less than 30 nmol/L, insufficiency to be between 30 and 50 nmol/L, and optimal vitamin D status to be above 50 nmol/L (32). The average requirement was determined to be 40 nmol/L, and is considered to be a desired level for population median, while 50 nmol/L represents the level at which most of the population will have their needs met (32). These cutoffs are based on promotion of optimal bone health in the healthy population (32). In cases of vitamin D intoxication, hypercalcemia can occur at 25OHD concentrations above 375500 nmol/L, and the cutoff for intoxication is conservatively set at 250 nmol/L (68). Children with Crohn’s disease represent a specific population for whom there exists no specific evidence-based 25OHD concentration cutoff.  As such, it is prudent to report the  proportions of children achieving different cutoffs, to enable comparison to other published research as well as to maximize the usefulness of this information as more evidence regarding optimal vitamin D status for this population emerges. 14  1.2.6.2  Determinants of Vitamin D Status Skin exposure to ultraviolet light is the primary determinant of vitamin D synthesis by the  endogenous pathway. Northern latitudes, winter season, sunscreen use, and increased amount of clothing worn are all associated with lower vitamin D status (50). Individuals who use tanning beds at least once per week have higher 25OHD concentrations and greater bone mineral density than those who do not (25OHD: 115.5 ± 8.0 vs. 60.3 ± 3.0 nmol/L respectively, p < 0.001) (69). Individuals with darker skin tones require greater UV-B exposure to produce equivalent vitamin D concentrations to those with lighter skin tones, because melanin acts as a natural sun screen (70). Latitude and season affect the solar zenith level, thereby impacting UVB radiation (71), such that at some latitudes (one estimate cited above 42.2°N) there is insufficient UVB radiation to promote cutaneous vitamin D production in the winter months (72). Without endogenous synthesis, individuals are dependent on vitamin D stores and exogenous sources of vitamin D. Obesity is associated with lower circulating levels of 25OHD from both dietary or endogenous pathways, likely because of greater sequestration of vitamin D in adipose cells (73).  1.3  Vitamin D Status of Children with Crohn’s Disease Several cross-sectional studies have assessed the vitamin D status of children with IBD  including Crohn’s disease (Table 1.2). Mean serum 25OHD levels reported in children with Crohn’s disease vary from 32.7 nmol/L in a cohort of newly diagnosed children in Germany, to 96.8 nmol/L reported in children with Crohn’s disease in Chicago (74,75). Such large variation in reported status reflects large heterogeneity in the subject populations including severity of disease, latitude, season, supplementation and fortification practices, as well as variation in assessment methodology.  Different 25OHD assessment methodologies were used including  high-performance liquid chromatography (76), radioimmunoassay (77), and various competitive 15  protein binding assays (75,78). Chemilluminescence immunoassays were the most commonly used technique (74,79–81). Various cutoffs for 25OHD were used to define vitamin D inadequacy in these studies, predominantly 25, ~37.5 and 50 nmol/L. The percentage of children with Crohn’s disease with 25OHD concentrations below the IOM cutoff for adequacy of 50 nmol/L ranged from 10 to 19% in American and Australian studies respectively (80,81), while higher rates of inadequacy were found using even lower cutoffs in other American and German studies (74,79). Considering the small sample sizes and large variability in subject characteristics, direct comparison to studies assessing the healthy population are not possible. However as a frame of reference, 14.1% of children aged 6-11 years old and 26.3% of children aged 12-19 years old, sampled as part of the Canadian Health Measures survey, have 25OHD concentrations below 50 nmol/L (82). Two studies have compared serum 25OHD concentrations in IBD pediatric patients to a non-IBD control group. One reported children with newly diagnosed IBD in Alberta to have a mean serum 25OHD concentrations of 66.6 nmol/L, which was significantly lower than the sexand age-matched controls mean of 81.7 nmol/L (p=0.044), although both were above the IOM cutoff for adequacy of 50 nmol/L (78). Another study of children with IBD in Helsinki, Finland, reported 25OHD concentrations to be higher in children with IBD than in healthy controls, although more children in the IBD group were receiving vitamin D supplements and a greater number were assessed in the summer months (76). When restricted to only those who were assessed in the winter, there was no difference between groups, (IBD: 43 (17 – 97) vs. non-IBD: 42.5 (17 – 82), p = 0.2) (76).  16  Table 1.2. Reported vitamin D status of children with inflammatory bowel disease Author  Year  Design  N  N per disease  Bechtold et al. (74)AM  2010  Crosssectional  143  98 with CD; 45 with UC  ElMatary et al. (78)AM  2011  Crosssectional  116  Gokhale et al. (75)AM  1998  Crosssectional  162  2012  Crosssectional  Laakso et al. (76)AM  160  39 with newly diagnosed CD; 21 with newly diagnosed UC; 56 matched controls 58 with CD; 37 with UC; 63 sibling controls 28 with CD; 49 with UC; 3 with colitis; 80 controls  Age (yrs)  Location  Vitamin D Supplementation  Mean Serum 25OHD (nmol/L) new CD: 32.7 (5.277.1) longer CD: 39.4 (10.0-128.3); new UC: 38.2 (8.0-49.9); longer UC: 34.4 (9.5-96.6)**  % Below Cutoff 20% of new CD and 17% of longer CD ≤ 25 nmol/L  6-21  Munich, GER  not specified  2.517  Edmonton, CAN  none  CD: 66.7 ± 27.3; UC: 56.9 ± 22.02; controls: 81.7 ± 15.4  not specified  5-18  Chicago, USA  not specified  CD: 96.8 ± 28.2; UC: 114.8 ± 32.2 p=0.008*  8.6% of CD < 25 nmol/L  5-20  Helsinki, FIN  48% of IBD patients, 21% of controls  IBD (n=41, winter only): 43 (17–97); control (n=76, winter only): 42.5 (17–82)  30% of IBD (summer and winter); 37% of controls (winter only) < 37.5 nmol/L  Levin et Retrospective mean Sydney, al. 2011 cross78 78 IBD = none IBD: 71.2 ± 26.5 AUS (80)AM sectional 12.6 Pappa et Retrospective 288 with CD; CD: 82.4 ± 30.0; UC: al. 2011 cross448 8-22 Boston, USA 59.40% 143 with UC 72.4 ± 27.5, p=0.006 (81)AM sectional Pappa et Cross94 with CD; CD: 49.9 ± 25.7; UC: al. 2006 130 8-22 Boston, USA 77% sectional 36 with UC 58.4 ± 29.2 (79)AM Sentongo Crosset al. 2002 112 112 with CD 5-22 Philadelphia, not specified sectional (77)AM USA CD: 65.9 ± 24.8 * Controls 25OHD levels not measured ** Standard deviation not given so range reported IBD = inflammatory bowel disease (includes Crohn's disease and ulcerative colitis, and may include indeterminate colitis) CD = Crohn's disease, UC = Ulcerative colitis  19% < 50 nmol/L 10.8% of CD ≤ 50 nmol/L 36% of CD were ≤ 37.44 nmol/mL 16% < 38 nmol/L  17  Differences in serum 25OHD concentrations between pediatric Crohn’s disease and ulcerative colitis patients have generally not been reported (74,79–81). In the largest crosssectional study of this population to date, conducted at the Children’s Hospital Boston, Pappa et al. reported mean serum 25OHD concentrations in 288 children with Crohn’s disease to be 82 nmol/L compared to 72 nmol/L in 143 children with ulcerative colitis (p=0.006). However, this difference lost significance once season of measurement, ethnicity, BMI z-score and vitamin D intake were controlled for in multivariate analysis (81). Factors found to be associated with the vitamin D status of children with Crohn’s disease are similar to known determinants of vitamin D status in the general population. Generally, serum 25OHD concentrations were lower in the winter months and in individuals with darker skin pigmentation (79–81). Where reported, vitamin D supplement use was positively correlated to 25OHD concentration (79,81). Contradictorily, BMI z-score was been both positively (79), and negatively (81) associated with 25OHD. In adults, increased disease activity has been associated with lower 25OHD concentrations (83), which may relate to poor absorption, decreased caloric intake, or some other disease-vitamin D interaction. In children, disease factors reported to be associated with lower vitamin D status include disease duration (79) and upper GItract involvement (79), while other studies have found no association between vitamin D status and disease location (80), or disease activity as assessed by the PCDAI (78). Overall, there is large variation in reported mean 25OHD concentrations in children with IBD, both between and within studies, which could indicate considerable prevalence of inadequate vitamin D status. It is not clear whether inadequacy is more prevalent in children with IBD than in the general population.  18  1.4  Vitamin D and Bone Health in Crohn’s Disease Poor bone health in individuals with Crohn’s disease is evidenced by their increased rates  of osteoporosis, osteopenia and incidence of fractures (12). Increased incidence rate ratios of fracture of 1.25 to 1.74 were reported in a Manitoba-based cohort study of 6027 adults with IBD compared to a sample of 60,270 non-IBD controls, depending on the site of injury (84). Decreased bone mineral density has been repeatedly observed in cross-sectional studies of children with inflammatory bowel disease when compared to healthy controls (74–76,85). This difference in bone health may be caused by both behavioural and genetic factors. Delayed pubertal timing, inflammation, poor nutritional intake and lack of weight-bearing physical activity may all contribute to decreased bone mineral density in children with chronic inflammatory disorders (86). It has long been reported that glucocorticoids can induce osteoporosis by modifying osteoblast and osteoclast activity (86). Glucocorticoid use has been associated with low bone mineral density in both adult Crohn’s disease patients (87) and children with IBD (75,88). For this reason glucocorticoid use is generally reserved for control of a flaring patient rather than long-term therapy. Unfortunately, impaired ability to achieve peak bone mass in young adulthood can increase individuals’ risk of osteoporosis and fracture later in life. In 2003, an extensive literature review conducted by the American Gastroenterological Association concluded that there exists a modest effect of IBD on bone mineral density, but that this is not a result of osteomalacia or vitamin D deficiency, due to a lack of observed relationship between the two (89). The review emphasized inflammation and corticosteroid use as the causes of low bone mineral density in IBD (89). Indeed, low vitamin D status often coincides with poor bone mineral density in children with Crohn’s disease, however no effect on bone mineral density as assessed by dual absorptiometry x-ray was observed in an open label experimental study of vitamin D and calcium supplementation in pediatric Crohn’s disease, reviewed in Section 1.5.3 19  (90). Reasons for lack of an observed effect could be the short duration of the study (12 months) relative to the method of bone health assessment. Further exploration of whether vitamin D supplementation could have an effect on a more short-term marker of bone health is warranted. In summary, although children with IBD frequently have lower bone mineral density than healthy controls, this may be as a result of the disease pathogenesis rather than vitamin D deficiency. To date, the only experimental study of the effect vitamin D supplementation on bone mineral density in pediatric Crohn’s disease did not show any benefit. 1.4.1  Bone Specific Alkaline Phosphatase in Children with Crohn’s Disease Bone biomarkers are indicators of the activity of osteoblasts and osteoclasts, the enzymes  responsible for the formation and resorption of bone (91). Bone specific alkaline phosphatase (BSAP), a membrane-bound osteoblast enzyme, is a widely used marker of bone formation (92). In adults, bone biomarkers are used to predict risk of osteoporosis and fractures, and to assess effectiveness of antiresorptive therapy (93). In children, concentrations of bone biomarkers fluctuate widely as a result of the bone modeling and epiphyseal growth that occurs in addition to the remodeling seen in adults, complicating interpretation of markers such as BSAP (93). In healthy controls (age 5 – 21 years), BSAP has been shown to be independently associated with sex, Tanner stage (stage of pubertal development), whole body bone mineral content, height velocity, and whole body bone mineral content accrual rates, explaining 80% of the variation in BSAP concentrations in multivariable linear regression (91). Children with Crohn’s disease have been found to have lower serum concentrations of BSAP and higher concentrations of urinary deoxypyridinoline, a marker of bone resorption, compared to healthy controls, even when values are adjusted for known determinants, suggesting poor bone mineral accrual in children with Crohn’s disease (91).  20  1.5 1.5.1  The Role of Vitamin D in Crohn’s Disease Epidemiologic Observations The hypothesis that a relationship exists between vitamin D and IBD arose from  epidemiological observation that disease prevalence and severity may be associated with sun exposure. Correlational evidence of this was particularly strong in the 1990s, and there have since been some conflicting reports. Crohn’s disease has long been reported to follow a “NorthSouth” gradient, with higher incidence and prevalence noted in Northern latitude countries such as Canada, the UK, and Northern Europe (4,94). This gradient has been quantified to find that incidence rates of inflammatory bowel disease are 40-80% higher in Northern latitudes (4,95), although there are exceptions to this association such as Australia, and the possibility of confounding variables, such as access to healthcare or hygiene practices, is considerable. Indeed, in the comprehensive epidemiological review presented in section 1.1.1, Molodecky et al. suggest that increasing rates of IBD in developing nations is associated with industrialization (6). A decreased incidence of Crohn’s disease has also been reported in outdoor workers compared to their indoor counterparts (96). In examination of data from the National Center for Health Statistics, the proportional mortality ratio (PMR) of people who died of Crohn’s disease or ulcerative colitis in the United States between 1991 and 1996 was significantly reduced amongst farmers, (PMR: 70, 95% confidence interval [CI]: 42–97), and laborers (71, 95% CI: 45–98), but also mining machine operators (31, 95% CI: 0 –74) who are not necessarily working outside (97). Additionally, increased Crohn’s disease relapse rates have been reported in the fall and winter months (98), although these findings were not repeated in another retrospective cohort investigation (99).  Such conflicting reports prevent definitive conclusions of whether a  correlation exists between sun exposure and Crohn’s disease. It could be that some studies have  21  not found evidence of this association because of flawed data collection or obscuring variables. Regardless, it has stimulated further research at the molecular, animal, and clinical levels. 1.5.2  Basic Research Basic research in animal models and in vitro is examining the potential mechanistic  interaction between vitamin D and IBD. Mutations in NOD2 (nucleotide-binding oligomerization domain-containing protein 2), a gene which encodes an antigen recognizing protein found in immune cells and intestinal epithelial cells which can trigger the proinflammatory pathway, have been associated with Crohn’s disease. Wang et al found 1,25(OH)2D3 to stimulate NOD2 mRNA production in monocytes and epithelial cells and to increase downstream protein synthesis, suggesting that vitamin D deficiency/insufficiency could play a causative role in prevalence of Crohn’s disease (100). It is important to note however, that normal circulating levels of 1,25(OH)2D3 in the body are approximately 1000 fold less than the concentrations examined by Wang. In mice, IBD models are induced by administration of dextran sodium sulfate (DSS) to drinking water or by genetically engineering susceptible populations such as interleukin-10 knockout (IL-10-/-) mice. Experiments examining the difference in vitamin D deficiency vs. sufficiency in colitis have used either VDR knockout (VDR-/-) approaches or dietary supplement approaches. VDR-/- mice are susceptible to DSS and have been observed to develop diarrhea, bleeding and weight loss eventually leading to death, while wildtype (WT) mice were mostly resistant to DSS-induced colitis (101,102). Similar observations of increased colitis severity in VDR-/- mice were recorded by Froicu et al. in two different murine models of IBD, including a CD4+/CD45RBhigh T cell transfer model and an IL-10-/- model (103). Researchers hypothesize that the VDR plays a role in maintaining intestinal epithelial integrity, due to in vitro observation  22  of enhanced tight junctions in 1,25(OH)2D3 treated cells (102). Impaired recovery is also seen in VDR-/- mice after removal of DSS compared to WT mice (101). IL-10-/- mice develop enterocolitis in an uncontrolled immune response to conventional microflora, while those raised in germ-free environments do not (104). Progression of IBD symptoms were compared in vitamin D deficient, vitamin D sufficient and vitamin D supplemented IL-10-/- mice by Cantorna et al (104). Diarrhea followed by wasting and mortality was seen in vitamin D-deficient IL-10-/- mice beginning at 7 weeks of age while vitamin Dsufficient IL-10-/- mice and vitamin D-deficient WT mice remained healthy up to 13 weeks (104). Small intestines of the vitamin D-deficient IL-10-/- mice were larger, heavier and more inflamed than those of either the vitamin D sufficient IL-10-/- or vitamin D deficient WT. Differences persisted even when restricted diets were imposed to rule out the confounding factor of weight loss. A decrease in enterocolitis was seen in vitamin D deficient IL-10-/- and vitamin D deficient WT mice supplemented with 1,25(OH)2D3 from age 7 weeks to 9 weeks. Small intestine weight was significantly heavier in non-supplemented vitamin D-deficient IL-10-/- mice than in nonsupplemented WT, supplemented WT, and supplemented vitamin D-deficient IL-10-/- mice (104). This indicates that vitamin D deficiency may play a role in the development of IBD, and may also impair recovery. While cell and animal studies provide a critical first step in elucidating biological plausibility of a link between vitamin D and IBD or Crohn’s Disease, unfortunately the results cannot be generalized to human subjects without further research. The IBD mice models produce similar symptoms and often respond to similar therapies as IBD in humans, but are induced by dietary or genetic manipulation while the true origin and progression of IBD in humans remains unconfirmed. Therefore there are inherent differences in the disease pathogenesis, in addition to the biological differences between mouse and human, which limit the generalizability of these 23  findings to the human subject. However this research does provide important evidence of a possible mechanistic interaction between vitamin D and IBD. 1.5.3  Prospective Human Research In addition to the vitamin D status of individuals with Crohn’s disease, summarized in  section 1.3, some limited experimental clinical research has attempted to determine the effect of vitamin D status or supplementation on the development or severity of Crohn’s disease. A recent study of the Nurses Health Study in the United States prospectively examined the development of ulcerative colitis and Crohn’s disease in relation to vitamin D status. Higher predicted values of 25OHD significantly reduced the risk of incident Crohn’s disease, and nonsignificantly reduced the risk of incident ulcerative colitis in the 72,719 adult women (105). 25OHD concentrations were predicted using a validated linear regression model based on factors such as dietary and supplemental vitamin D intake, exposure to sunlight, race, body mass index, and regional ultraviolet-B radiation intensity and adjusted for season of blood draw (105). Study findings indicate that lower vitamin D status is not only associated with development of Crohn’s disease, but in fact it precedes it, and therefore low vitamin D status could be a Crohn’s disease risk factor. In a randomized controlled trial evaluating the effect of vitamin D supplementation on Crohn’s disease activity, 94 adults with Crohn’s disease who were in remission (Crohn’s Disease Activity Index (CDAI) < 150) were recruited and assigned to either a vitamin D supplement of 1200 IU/day, or a placebo (106). There was a trend towards a reduced rate of relapse (CDAI > 150 or an increase in CDAI > 70) in the vitamin D-treated group compared to the placebo group (13% vs. 29%), although it was not found to be statistically significant (p=0.06) (106). Despite this failure to reach statistical significance, the trend is a promising one that merits further investigation. 24  In children, Benchimol et al. conducted an open label prospective trial on the effect of calcium and vitamin D supplementation in 72 children with IBD and low bone mineral density (90). After 12 months of supplementation there was no difference in change in height z scores or bone mineral density between a calcium supplemented group, a calcium and vitamin D supplemented group, and a control group (90). This may have been too short a duration to observe a difference in bone mineral density between groups as a result of supplementation. Additionally, this study was limited by its open label design and the mismatched control group, which had higher baseline bone mineral density than the two intervention groups. Most recently, Pappa et al. conducted a randomized controlled trial of the effect of 6 weeks of vitamin D supplementation (either 2000 IU vitamin D2 daily, 2000 IU vitamin D3 daily, or 50,000 vitamin D2 weekly) on vitamin D status of 71 children with IBD (107). Mean baseline 25OHD concentrations were low, between 36.7 and 40.2 nmol/L, increasing to between 64.1 and 101.8 depending on dose received. Daily vitamin D3 and weekly vitamin D2 supplementation were significantly more effective (p = 0.03 and p = 0.0004) at raising serum 25OHD concentrations than the daily vitamin D2 supplement (mean increase in serum 25OHD was 41, 63 and 23 nmol/L respectively) (107). Seventy-five percent of subjects receiving 2000 IU vitamin D2 daily achieved serum 25OHD concentrations >50 nmol/L, while 95% achieved this cutoff in the other two treatment groups (p = 0.14). Twenty-five, 38, and 75 % of subjects achieved 25OHD concentrations > 80 nmol/L in the 2000 IU vitamin D2 daily, 2000 IU vitamin D3 daily, or 50,000 vitamin D2 weekly groups respectively (p = 0.004).  There was no significant  difference in safety marker concentrations of serum calcium, serum phosphorus, or urinary calcium/creatinine ratio between groups. This study provides important information to clinicians of the safety and efficacy of vitamin D deficiency treatment over the short-term, however cannot inform long-term or ongoing supplement regimens.  To date there are no long-term 25  supplementation trials examining the efficacy and safety of lower dose vitamin D supplementation in pediatric IBD.  1.6  Summary of Evidence and Gaps to be Addressed in this Thesis Crohn’s disease is a chronic, recurring disorder marked by inflammation of the  gastrointestinal tract. Crohn’s disease in children can result in nutritional deficiencies, inadequate weight gain, growth failure, and a decreased quality of life. Although remission can be achieved through medical and/or surgical therapies, risk of relapse remains. Vitamin D is a fat soluble prohormone that can be obtained through the diet or produced endogenously in the skin when exposed to ultraviolet light. The active form of vitamin D, 1,25(OH)2D binds to the vitamin D receptor found in cells throughout the body, to stimulate diverse actions. These range from maintaining serum calcium homeostasis, which is critical to promotion of adequate bone mineral density, to immune system regulation. Particularly relevant to Crohn’s disease, 1,25(OH)2D3 has been found to inhibit Th1 and Th17 action (61–63). Vitamin D status is best assessed by measurement of serum 25OHD concentrations. Cutoffs for adequacy are controversial. Currently Health Canada advises that concentrations below 30 nmol/L represent deficiency, 30-50 nmol/L represents insufficiency, and concentrations above 50 nmol/L represent optimal vitamin D status, however these are based on promotion of optimal bone health in the healthy population (32,64). No specific cutoffs for children with Crohn’s disease exist, and a variety of cutoffs are used within the scientific research community, including some as high as 75 or 80 nmol/L (80,107). Vitamin D status of children with Crohn’s disease has been assessed in a variety of settings, and results vary largely between and within reports. Mean serum 25OHD concentrations reported in children with Crohn’s disease vary from 32.7 nmol/L in a cohort of 26  newly diagnosed children in Germany, to 96.8 nmol/L reported in children with Crohn’s disease in Chicago (74,75). Vitamin D status in children with Crohn’s disease is partially determined by season of blood draw, skin tone of the subject, and supplement use, and may also be associated to disease duration. Decreased bone mineral density has been repeatedly observed in children with IBD when compared to healthy controls, and is believed to be a result of behavioural determinants (diet, weight-bearing physical activity), genetic factors, inflammation, pubertal timing and glucocorticoid use. Although low vitamin D status often coincides with poor bone mineral density in children with Crohn’s disease, vitamin D supplementation showed no effect on bone mineral density as assessed by dual absorptiometry x-ray in a 12 month open label trial (90). Effect of supplementation on a more short term marker of bone health, for example a marker of bone formation, BSAP, has not been assessed. Epidemiologic observations relating sun exposure to Crohn’s disease prevalence and severity lead to the hypothesis that vitamin D may play a role in disease development or progression, and has stimulated in vitro, animal and clinical research. Vitamin D deficiency has been shown to aggravate colitis in IL-10-/- mice compared to vitamin D sufficient counterparts, and promisingly, 1,25(OH)2D3 supplementation has been shown to improve colitis symptoms in these mice (104). In humans, low predicted 25OHD status has been shown to precede Crohn’s disease development in a large American cohort study (105). Experimentally, vitamin D supplementation has not shown significant impact on either disease activity or bone mineral density in individuals with Crohn’s disease, however both deserve further consideration due to limitations in the research that has been conducted. In the only randomized controlled trial of vitamin D supplementation in children with Crohn’s disease to date, 2000 IU of vitamin D3 per 27  day or 50,000 IU vitamin D2 per week were established as safe, effective supplement regimens over the short term (6 weeks) to promote adequate vitamin D status (25OHD >50 nmol/L) (107). Further rigorous experimental research is needed to establish safe and effective vitamin D supplementation recommendations for children with Crohn’s disease over the long term, as well as to further elucidate the effects of supplementation on indicators of disease activity and bone health. Considering this, the following objectives were developed for this research project.  1.7  Research Objectives  Primary objective: 1. To determine whether a vitamin D3 supplement dose of 2000 IU/day will achieve a higher prevalence of serum 25OHD concentrations greater than 50 or 75 nmol/L compared to a control dose of 400 IU/day, in children with quiescent Crohn’s disease. Secondary (exploratory) objectives: 2. To evaluate the safety of two supplement regimens through monitoring of serum calcium and phosphate concentrations and urinary calcium to creatinine ratio. 3. To determine if children with Crohn’s disease receiving 2000 IU/day vitamin D have greater concentrations of bone specific alkaline phosphatase, a marker of bone formation, compared to those receiving 400 IU/day. 4. To determine if children with Crohn’s disease receiving 2000 IU/day vitamin D are more likely to achieve sustained remission, as assessed by the Pediatric Crohn’s Disease Activity Index, than those receiving 400 IU/day.  28  Chapter 2: Research Methods 2.1  Purpose Outcomes from this study will help inform health practitioners and caregivers of  appropriate, safe, and effective vitamin D supplementation recommendations for children with Crohn’s disease, in order to promote adequate vitamin D status in this vulnerable population.  2.2  Overview A double-blind randomized controlled study design was used for this project. It was a 6-  month supplementation trial comparing two treatments arms: 400 IU vitamin D3 vs. 2000 IU vitamin D3. Baseline, 3-month, and 6-month study visits were completed with all subjects. Two sites participated in this study: British Columbia (BC) Children’s Hospital in Vancouver and McMaster Children’s Hospital in Hamilton. The same protocol and study documents were used at both sites.  2.3  Sample Size Sample size was originally calculated to be able to determine a difference in proportions  of subjects achieving serum 25OHD concentrations greater than the cutoff of 75 nmol/L. To detect a difference of 25%, using 80% power, an α = 0.05, and a two-sided test design, 58 subjects were needed per group. In addition, to account for attrition, an additional 20% needed to be recruited. In total 140 subjects were needed.  2.4  Participant Selection and Recruitment The University of British Columbia Children and Women’s Clinical Research Ethics  Board approved this study protocol (identifier: H10-01391).  The Hamilton Health 29  Sciences/Faculty of Health Sciences Research Ethics Board approved this study at the McMaster Children’s Hospital site (identifier: 10-393).  The study was also registered online with  clinicaltrials.gov (identifier: NCT01187459), a publicly accessible online registry of current and completed clinical trials, founded jointly by the National Institute of Health and the Food and Drug Administration in the United States. To participate in the study, subjects were required to meet the following eligibility criteria of being: •  Diagnosed with Crohn’s disease  •  Between 8-18 years old  •  In remission (as assessed by a PCDAI < 10) Subjects were excluded if they were currently being prescribed glucocorticoids, or if they  had been taking them within the previous 6 weeks, or if they were consuming more than 400 IU of vitamin D supplements per day. The Master’s student identified patients with Crohn’s disease who were potentially eligible for the study by reviewing patient charts prior to their visits to the outpatient hospital Gastroenterology Clinic or the Medical Day Unit (for those children receiving infliximab infusions). Letters of contact (Appendix A.1) were mailed to patients and their families, which were followed up with a phone call to the family, to further determine eligibility and level of interest.  The consent form was emailed to interested families for their review. Prior to their  clinic visit, the Master’s student or research assistant (will be referred to as the Master’s student from here onward) would approach interested families and further explain the study protocol. Upon completion of the clinic visit, the Master’s student would confirm with the pediatric gastroenterologist that the patient was in remission and eligible for the study. In these cases, so  30  long as the patient had previously reviewed the consent form, the Master’s student was able to obtain written informed consent/assent (Appendices A.2, A.3) as appropriate from the child and/or guardian, and proceed with the first study visit.  The same recruitment procedure was  used at the Hamilton Health Sciences site as at the Vancouver site. Subjects were provided with $50 in gift certificates to Future Shop or Chapters as remuneration for participating in the study.  2.5  Procedures Subjects completed three study visits: one at enrollment, one at the midpoint of the study  (3 months), and one at the endpoint of the study (6 months). When possible, visits were scheduled at the same time as gastroenterology follow-up appointments, however generally at least one visit required the participant to make an extra trip to the hospital for study purposes. At enrollment, subjects were assigned a study ID, either a CDV (Crohn’s Disease Vancouver) or CDH (Crohn’s Disease Hamilton) code followed by a three digit number that was assigned ordinally, beginning with 001. Baseline information such as participant age, ethnicity, medication and supplement use was questioned and recorded. Subjects consuming a vitamin D supplement (less than 400 IU/day) were asked to discontinue that supplement in favor of the provided study supplement. A specially designed multivitamin, not containing vitamin D, was offered to study subjects. For further description of the study supplements and randomization, see section 2.6. Dietary vitamin D and calcium intake were assessed at the 3-month visit by a validated food frequency questionnaire (FFQ), see section 2.7. At each visit, nursing staff measured subjects’ height and weight. Blood and spot urine samples were collected by the hospital laboratory staff. Subject medication and supplement use was questioned and recorded by the Master’s student. Medication dosing and PCDAI score were 31  confirmed through review of the patient’s medical chart. The PCDAI (Appendix B), introduced in Section 1.1.3, is part of routine care. Patients’ disease activity is assessed and scored by the responsible gastroenterologist, based on a compilation of biochemical and physical assessments as well as patient-reported history. Each item is scored on a 3-point scale of 0, 5 or 10, with the exception of hematocrit and erythrocyte sedimentation rate (ESR), which are scored a 0, 2.5 or 5 (9). Low scores indicate less disease activity. Generally, a total score of less than 10 signifies the patient has inactive disease, a score 11-30 signifies mild disease, and a score 31-100 signifies moderate/severe disease (108).  2.6  Dietary Supplements, Randomization, Adherence Study supplements provided to participants included the vitamin D3 supplement (bound  with magnesium stearate and microcrystalline cellulose) and a chewable children’s multivitamin (Table 2.1). The supplements were manufactured and packaged for study purposes by Natural Factors of Nutritional Companies. A letter of no objection was provided by Health Canada. Supplement doses were validated internally on numerous occasions and externally by Heartland Assays LLC in March 2012 (Table 2.2). Dosing amounts of the two treatment arms, 400 IU vs. 2000 IU, were decided prior to the IOM and Health Canada’s revised vitamin D intake guidelines set in November 2010. At the time of study design, 400 IU was the vitamin D Adequate Intake and 2000 IU was the UL for children. Since then, the RDA has been increased to 600 IU in children, and the UL has been raised to 3000 IU for eight year olds, and 4000 IU for children nine years and older (32). The study dosing regimen remained an appropriate dosage as most children obtain additional vitamin D through their diet and/or sun exposure. Moreover, since many children with Crohn’s disease already take a daily multivitamin, which generally contains 400 IU vitamin D, this was kept as the control dose so as to not induce a decrease in serum 25OHD 32  concentrations in subjects. No placebo was used in this study, as it could be considered unethical to deny a vitamin D supplement to participants. Table 2.1. Ingredient list of the study multivitamin Ingredient Vitamins Beta carotene (provitamin A) Vitamin B1 (thiamine mononitrate) Vitamin B2 (riboflavin) Niacinamide Vitamin B6 (pyridoxine hydrochloride) Vitamin B12 (cyanocobalamin) D’Pantothenic acid (calcium pantothenate) Folic acid Biotin Vitamin C (sodium ascorbate/ascorbic acid) Vitamin E (d-alpha tocopheryl acid succinate) Para amino benzoic acid (PABA) Citrus bioflavonoids (Citrus sinensis, C. limonum) Lipotropic Factors Choline bitartrate Inositol Minerals Calcium (calcium citrate) Magnesium (magnesium oxide) Iron (ferrous fumarate) Potassium (potassium citrate) Zinc (zinc citrate) Manganese (manganese gluconate)  Quantity 5000 IU 5 mg 5 mg 10 mg 5 mg 10 mcg 10 mg 0.2 mg 10 mcg 100 mg 25 IU 5 mg 5 mg  Table 2.2. Vitamin D supplement dosage quality control (IU) 400 IU Tablet Natural Factors Inc. April 20, 2010 488 January 17, 2011 473 August 3, 2011 463 Heartland Assays LLC March 27, 2012 484  5 mg 5 mg 65 mg 25 mg 5 mg 5 mg 2 mg 1 mg  2000 IU Tablet 2490 2523 2209 2379  33  Study supplements were coded numerically by lot number, so that the dosages were unknown to the Master’s student, research assistants, investigators, responsible physician, and subjects themselves. A statistician performed the randomization, assigning each subject ID to one of the study supplement lot numbers. Subjects were not given special instructions regarding the timing of consumption of their study vitamins, other than to try to consume the supplement at the same time each day, so as to incorporate the habit into their daily routine. Supplements were distributed in sealed bottles containing 100 pills, to subjects at baseline and 3-months. Subjects were asked to return remaining pills with the pill bottles for weighing at the follow-up visits of 3-months and 6-months. Number of pills consumed was divided by days between visits to determine subject adherence.  2.7  Dietary Assessment Vitamin D and calcium dietary intake was assessed at the second study visit by a  validated food frequency questionnaire (FFQ). Parents were asked to complete the questionnaire for young or distracted participants, while older subjects were able to complete the questionnaire themselves.  Developed by Whiting et al., the FFQ is a 38-item questionnaire that asks  participants to quantify both the frequency and portion size of food consumed over the previous month (Appendix C). The FFQ has been validated in young Canadian adults of diverse ancestry and found to correlate well with a 7-day food diary (r = 0.529, p<0.001) (82). Coded FFQs were mailed to the developers at the University of Saskatchewan College of Pharmacy and Nutrition for analysis. Calcium and vitamin D content for each food item in the FFQ was determined using an EHSA Food Processor (Version 8.0, EHSA Research, Ore), which included the 1997 Canadian Nutrient File from Health Canada. 34  2.8  Biochemical Measures  2.8.1  Collection and Processing of Blood and Urine Samples Subjects were asked to provide a mid-stream spot urine sample and blood sample at each  study visit. Blood was drawn by venipuncture by hospital phlebotomists at the hospital blood collection laboratory or by nurses of the Medical Day Unit (for children receiving infliximab infusions) according to facility protocols into a 10 mL glass, silicone-coated interior, vacutainer, without additives. The silicone coating reduces cells adherence to the tube walls. Unless the subject had completed a blood test within the past week as part of standard care, an additional 2 mL of blood were collected and kept by the pathology department for immediate analysis (complete blood count and ESR). Once collected, the Master’s student allowed the 10 mL sample of blood to clot for 45 minutes (but within 60 minutes), before running it in the centrifuge at 3000 rotations per minute for 10 minutes, to separate the serum from the red blood cells. At the BC Children’s Hospital site, serum was pipetted into three labeled 2 mL plastic tubes and urine was pipetted into five 2 mL plastic tubes by the Master’s student and all samples were frozen in lab freezers of -80°C. At the McMaster Children’s Hospital, the Laboratory Research Coordinator performed the same procedure and stored three samples of serum and two samples of urine per subject. 2.8.2  Analysis of Serum and Urine Samples Both 25OHD and BSAP analyses for all samples were conducted by the laboratory of Dr.  Hope Weiler at McGill University in the School of Dietetics and Human Nutrition. Four separate batches of analyses were conducted, in November 2011, April 2012, August 2012, and November 2012. Samples, which had not undergone any freeze-thaw cycles, were removed from the -80°C  35  storage freezers and shipped by FedEx on dry ice to Dr. Weiler’s laboratory. All samples arrived in good time and remained frozen. The primary outcome measure, serum 25OHD concentration, was assessed using the DiaSorin LIAISON 25-OH Vitamin D Total Assay (DiaSorin Inc, Stillwater, MN, USA), a competitive chemiluminescence immunoassay. The assay detects both 25OHD2 and 25OHD3, and is therefore a good assessment of overall vitamin D status in an individual. Normalized to 25OHD3, the cross-reactivity of other vitamin D metabolites vitamin D2, vitamin D3 and 3-epi25OHD3 are all <1%. Even though 1,25(OH)2D2 and 1,25(OH)2D3 are 40% and 17% crossreactive, these metabolites generally circulate at low levels and so do not skew the assessment. The assay measurement range is 10-374 nmol/L. The intra-assay coefficient of variation (CV)% was calculated from repeated measurement of a pooled plasma sample used as a laboratory reference range for healthy adults and from DiaSorin high and low controls. The 25OHD intraassay CV% (Table 2.3) ranged from as low as 1.3% to as high as 10.8%.  The accuracy was  calculated using the mid-range manufacturer specifications for DiaSorin low controls and for DiaSorin high controls and ranged from 84 to 112%. Table 2.3. Summary of 25OHD intra-assay coefficient of variation (CV%) Date 25OHD Analysis CV% BSAP Analysis CV% 04-Dec-12 1.3 - 5.5 3.6 31-Oct-12 1.3 - 10.8 3.4 - 8.4 26-Apr-12 8.0 13.0 – 17.0 10-Nov-11 3.6 - 6.0 1.3 - 10.3 BSAP is a biomarker of bone formation that can be measured in serum, and unlike many other bone biomarkers, is not subject to circadian variability (91). BSAP was assessed using the DiaSorin LIAISON BAP Ostase chemiluminescence immunoassay. The assay measurement  36  range is 1.5-120 ug/L. The analytical sensitivity is 0.1 ug/L, while the functional sensitivity is 1.5 ug/L. Intra-assay CV% (Table 2.3) ranged from 1.3 to 17, and accuracy ranged from 84 to 99%. Analyses conducted by the respective hospital laboratories included hematocrit, ESR, serum calcium, phosphate, creatinine and albumin, and urinary calcium, phosphate, and creatinine concentrations. All tests were completed by standardized laboratory procedure using Johnson & Johnson, Ortho-Clinical Diagnostics VITROS© 5600 System laboratory equipment at BC Children’s Hospital and the Roche Modular Analytics system at McMaster Children’s Hospital. Normal ranges used are presented in Table 2.4. PCDAI cutoffs are also presented for hematocrit, albumin and ESR as these are all biochemical indices used in the index. Serum calcium, serum phosphate and urinary calcium/creatinine ratios are safety markers for hypervitaminosis D. They are commonly used in clinical vitamin D supplementation research to indicate hypercalcemia, the side effect of hypervitaminosis D (107). The BC Children and Women’s Health Centre of British Columbia Department of Pathology and Laboratory Medicine uses age specific cutoffs for serum calcium and phosphate concentrations, and a level 0.7 mmol/mmol as the cutoff for normal urinary calcium/creatinine ratio. A calcium/creatinine ratio cutoff of 0.6 mmol/mmol has also been used in some research (107).  37  Table 2.4. Normal ranges for biochemical measures Test Normal range PCDAI cutoff (score assigned)  Hematocrit  6yrs: 0.35-0.43; 12yrs: 0.35-0.44  <10yrs: >33 (0), 28-32 (2.5), <28 (5) 11-19yrs F: >34 (0), 29-33(2.5), <29 (5), 11-14yrs M: >35 (0), 30-34 (2.5), <30 (5) 15-19yrs M: >37 (0), 32-36 (2.5), <32 (5)  ESR  F: 0-25; M: 0-11 mm/hr  <20 (0), 20-50 (2.5), >50 (5)  Serum Albumin  37-56 g/L  >35 (0), 31-34 (5), <30 (10)  Serum Phosphate  7 yrs: 1.19-1.81 12 yrs: 1.07-1.74; 14yrs: 0.94-1.74; 16 yrs: 0.87-1.52 mmol/L  n/a  Serum Calcium  4yrs: 2.20 - 2.52; 10 yrs: 2.22-2.52; 12yrs: 2.20-2.64; 14yrs: 2.30-2.67; 16yrs: 2.22-2.67 mmol/L  n/a  Urinary Calcium: <0.7 mmol/mmol n/a Creatinine Ratio Cutoffs from the Department of Pathology and Laboratory Medicine Internal Policy/Procedure at the Children's and Women's Health Centre of British Columbia  2.9  Data Analysis Statistical analyses were performed using SPSS Statistics 20.00 for Macintosh (SPSS Inc.,  Chicago, IL 2012). Data was assessed for normality using histograms and found to be normal. PCDAI and BSAP were not normally distributed. Baseline characteristics were summarized by  38  number of subjects and percentage for categorical variables and by reporting means and standard deviation (SD) for numerical variables. Outcomes were reported using an intent to treat model (ITT). In ITT analysis, last recorded values are carried forward to the endpoint of withdrawn subjects. This is done in order to account for any systematic differences that may exist between subjects who withdraw from a study and those who remain enrolled. 25OHD outcomes were also analyzed using an as-treated model (where data is analyzed without any values carried forward), and by those who were reportedly 80% adherent with the supplements, for comparison to the ITT outcomes. The primary objective, to determine the difference in number of subjects achieving 25OHD concentration cutoffs per group, was assessed by chi-square test. A cutoff level of 25 nmol/L was used to define deficiency (66). A cutoff of 50 nmol/L was used to define vitamin D adequacy, in accordance with the IOM’s recommendations for healthy children and adults (32). Percentage of subjects achieving 75 nmol/L was also reported to enable comparison with other published research in this population. 25OHD concentrations were also evaluated by comparing the difference in means between groups at each time point, adjusting for baseline values in a general linear model. Baseline 25OHD concentrations were also examined for effect of known determinants such as season, ethnicity, vitamin D intake and age, using a general linear model. The means of secondary outcomes including PCDAI, BSAP, and safety markers such as serum calcium, serum phosphate and urinary calcium/creatinine ratio were all compared between groups, at baseline, 3months and 6-months, using one-way ANOVA. Additionally, difference between groups in mean BSAP concentrations adjusted for baseline values in a general linear model, was assessed. A significance level of 0.05 was used for all analyses.  39  Chapter 3: Results 3.1  Recruitment and Randomization Eighty-three children were recruited and enrolled in the study, between November 2010  and February 2012. Fifty-three subjects were enrolled at the Vancouver site and 30 at the Hamilton site. Protocol completion and withdrawals are summarized in Figure 3.1. Seventythree subjects completed the 3-month study visit, and 69 completed the 6-month study visit. Of the 14 subjects who did not complete the protocol, 5 were lost to follow-up, 3 were selfreportedly non-adherent and asked to be withdrawn, 1 experienced a serious flare-up, 1 became uncomfortable not knowing the supplement dosage, 1 reported travel distance as the reason for withdrawal, 1 reported low energy levels and 1 gave no explanation for withdrawal. One subject’s 6-month study data had to be excluded due to a protocol error, wherein the subject had be given the wrong supplement dosage. One subject did not complete the baseline bloodwork and subsequently withdrew, therefore the maximum number of blood or urine concentration values at any timepoint is 82. Two subjects skipped their 3-month study visit but were mailed their study supplements and able to complete the 6-month study visit. Forty subjects (14 from the Hamilton site) were randomized to the 400 IU arm, and 43 (16 from the Hamilton site) to the 2000 IU arm.  40  Assessed for eligibility and enrolled, n = 83  Baseline visit Randomized to 2000 IU arm, n = 43 -  3 withdrew -  2 lost to followup -  1 skipped appointment Completed 3-month follow-up, n = 37 -  2 lost to followup -  1 excluded due to protocol error Completed 6-month follow-up, n = 35  Baseline visit Randomized to 400 IU arm, n = 40 -  3 withdrew -  1 skipped appointment Completed 3-month follow-up, n = 36 -  2 withdrew -  1 lost to followup Completed 6-month follow-up, n = 34  Figure 3.1. Subject Flow Diagram.  3.2  Adherence Adherence to the supplement regimen as assessed by weighing returned supplement  bottles is summarized in Table 3.1. Adherence information was available for 63 subjects, of which 62% had greater than 80% adherence. Fourteen subjects had less than 70% adherence, 10 had 70 – 80% adherence, 31 had 80 – 100% adherence, and 8 had greater than 100% adherence, indicating error in the manner of adherence assessment (elaborated upon in discussion). When categories were collapsed to either <80% or >80% adherence, there was no significant difference between groups at any time point (not shown). Groups needed to be collapsed due to low number of observed and expected case counts. 41  Table 3.1 Adherence as reported by subjects, n (%) < 70% 70 - 80% All, n = 63 14 (22) 10 (16) Treatment 400 IU, n = 29 5 (17) 4 (14) 2000 IU, n = 34 9 (27) 6 (18)  3.3  80 - 100% 31 (49)  >100 % 8 (13)  14 (48) 17 (50)  6 (21) 2 (6)  Baseline Characteristics Baseline subject characteristics are summarized in Table 3.2. Mean age was 14.3 (±  standard of deviation [SD] = 2.3) years. Slightly less than half were female, 46%. The majority of subjects were white, 78%. When subjects reported mixed ethnicity, they were reported as the non-white ethnicity. Of the 19 subjects who were not white, 10 were of South-Asian descent, 4 were black or half-black, 2 were Asian or half-Asian, 2 were of Caribbean descent, and one was of Austronesian background. Mean weight was 48.7 (± 12.6) kg, and mean height was 158 (± 13) cm. The lowest percentage of subjects were recruited in the summer months of June 21st to September 20th, 8%, while 18, 33 and 41% of subjects were recruited in the spring, fall and winter respectively. The majority of subjects were using immunomodulator medications such as azathioprine or methotrexate, and some subjects took these concurrently with 5-aminosalicylates or biologic therapies. There was no significant difference in any of the baseline characteristics between treatment arms.  42  Table 3.2 Subject baseline characteristics Mean ± SD or N (%) Characteristic Age (years) Female Ethnicity, Caucasian Weight Height Season of enrollment Spring (Mar 21 - Jun 20) Summer (Jun 21 - Sep 20) Fall (Sep 21 - Dec 20) Winter (Dec 21 - Mar 20) Medications 5-ASA users Immunomodulator users Biologics (infliximab and/or adalimumab) users Taking supplement containing vitamin D Mean vitamin D intake (IU) from supplement in supplement users alone, n = 26, 14, 12 Dietary Vitamin D intake (IU), n = 72, 33, 39a Dietary Calcium intake, n = 72, 33, 39a * N = 83, 40, 43 unless otherwise specified. a Measured at study midpoint. 3.3.1  All subjects, n = 83 14.3 ± 2.3 38 (45.8) 65 (78) 48.7 ± 12.6 158 ± 13  400 IU, n = 40 14.0 ± 2.4 19 (46.3) 31 (78) 48.7 ± 13 157 ± 13  2000 IU, n = 43 14.5 ± 2.1 19 (44.2) 34 (79) 48.6 ± 12.4 159 ± 13  15 (18) 7 (8) 27 (33) 34 (41)  7 (18) 3 (8) 14 (35) 16 (40)  8 (19) 4 (9) 13 (30) 18 (42)  30 (36) 47 (57)  17 (43) 21 (53)  13 (30) 26 (61)  25 (30) 26 (31)  11 (28) 14 (35)  14 (33) 12 (28)  420 ± 141  414 ± 151  428 ± 135  208 ± 134 762 ± 448  189 ± 123 720 ± 386  224 ± 141 798 ± 497  Dietary Intake Thirty-one percent of subjects were consuming a supplement containing vitamin D prior  to study enrollment. Some subjects reported taking 1000 IU supplement 1-2 times per week in which case their intake per day was calculated based on their reported consumption. The mean vitamin D intake from supplement source was 420 (± 141) IU/d. Mean dietary vitamin D intake of the 72 subjects who completed the FFQ was 208 (± 134) IU/d. Including supplement sources, at baseline the mean intake would have been  43  approximately 638 (± 190) IU/d for supplement users and 203 (± 144) IU/d for non-supplement users. Mean calcium intake was 762 (± 448) mg. 3.3.2  Baseline 25OHD by Participant Characteristic Difference in baseline 25OHD concentrations was also assessed by known determinants  in a general linear model. Age and vitamin D intake (from supplement and diet) were significant covariates. Season of assessment was not a significant factor, when assessed separately or when collapsed into groups of summer and fall vs. winter and spring. Serum 25OHD concentrations were 15 nmol/L higher in white and than in non-white participants (p = 0.002) (Table 3.3). In total, these known determinants only accounted for 30% of the variation in 25OHD concentrations in subjects (adjusted R squared = 0.302).  Table 3.3. Serum 25OHD concentration, by predictor Estimated difference in Marginal Mean Predictor 25OHD, nmol/L (95% CI) (95% CI) p value Ethnicity White, n = 64 15.3 (5.6 - 24.9) 62.4 (57.8 - 67.0) 0.002 Non-white, n = 18 0 47.1 (38.5 - 55.8) Season of enrollment Summer/Fall (Jun 21 - Dec 20) -4.6 56.5 (49.1 - 64.0) 0.414 Winter/Spring (Dec 21 - Jun 20) 0 53.0 (47.4 - 58.5) Baseline 25OHD concentrations adjusted for covariates of age (p = 0.018) and vitamin D intake at baseline (including diet and supplement, p = 0.001). Model adjusted R squared = 0.302.  44  3.4  Proportion of Subjects Achieving 25OHD Cutoffs Number of subjects achieving the serum 25OHD concentration cutoffs at each time point  was reported using ITT (Table 3.4) and as-treated (Table 3.5) models as well as by those who were reportedly 80% adherent (Table 3.6). Only two subjects were below the deficiency cutoff of 25 nmol/L at baseline, and none were deficient at the 3-month and 6-month follow-up study visits. The number of subjects above the 50 nmol/L cutoff at baseline was 55 (67%). In intentto-treat analysis, the majority of subjects were able to achieve serum 25OHD concentrations of 50 nmol/L or greater by 6 months in both the 400 IU group (35 (87%)), and the 2000 IU (37 (88%)) group. Nor was there a difference between groups at 3 months. In as-treated analysis, the number of subjects above the 50 nmol/L cutoff for adequacy increased more dramatically in the 2000 IU group by the 3-month visit (400 IU: 79% vs. 2000 IU: 97%, p = 0.021), however by 6months there was no significant difference between groups. In the 80% adherent group, there appeared to be no difference in number of subjects above 50 nmol/L by supplement dose at 3 or 6 months, however the sample size is quite small. In the majority, 65 (79%), of subjects, serum 25OHD concentrations were below the cutoff of 75 nmol/L at baseline. In ITT analysis, there was no significant difference in number of subjects above the 75 nmol/L cutoff at 3 months, however by 6-months the difference was pronounced, 14 (35%) vs. 31 (74%) in the 400 and 2000 IU groups respectively (p < 0.001). A similar trend was seen in the 80% adherent subgroup, while in the as-treated analyses there was a significant difference between the dose groups at both the 3-month and 6-month assessments.  45  Table 3.4:Proportion of subjects achieving 25OHD cutoffs, ITT Baseline 400 IU 2000 IU 400 IU Outcome (n=40) (n=42) p value (n=40) Serum 25OHD (nmol/L), mean ± SD < 25 nmol/L, N (%) 0 (0) 2 (5) 0.492* 0 (0) ≥ 50 nmol/L, N (%) 31 (77) 24 (57) 0.05 32 (80) ≥ 75 nmol/L, N (%) 11 (27) 6 (14) 0.177 16 (40) * Expected cell count less than 5, Fisher’s Exact Test used Table 3.5: Proportion of subjects achieving 25OHD cutoffs, as treated Baseline 400 IU 2000 IU 400 IU Outcome (n=40) (n=42) p value (n=34) Serum 25OHD (nmol/L), mean ± SD < 25 nmol/L, N (%) 0 (0) 2 (5) 0.492* 0 (0) ≥ 50 nmol/L, N (%) 31 (77) 26 (57) 0.05 27 (79) ≥ 75 nmol/L, N (%) 29 (73) 6 (14) 0.177 14 (41) *Expected cell count less than 5, Fisher’s Exact Test used  3 months 2000 IU (n=42)  p value  0 (0) 37 (88) 25 (60)  n/a 0.316 0.077  400 IU (n=40)  6 months 2000 IU (n=42)  p value  0 (0) 35 (87) 14 (35)  0 (0) 37 (88) 31 (74)  n/a 0.934 <0.001  p value  n/a 1.000* <0.001  3 months 2000 IU (n =35)  p value  400 IU (n=34)  6 months 2000 IU (n=34)  0 (0) 34 (97) 24 (69)  n/a 0.028* 0.022  0 (0) 31 (91) 12 (35)  0 (0) 32 (94) 27 (79)  46  Table 3.6: Proportion of subjects achieving 25OHD, 80% adherence Baseline 2000 400 IU IU 400 IU Outcome (n=20) (n=19) p value (n=20) Serum 25OHD (nmol/L), mean ± SD < 25 nmol/L, N (%) 0 (0) 0 (0) n/a 0 (0) ≥ 50 nmol/L, N (%) 17 (85) 18 (58) 0.082* 17 (85) ≥ 75 nmol/L, N (%) 5 (25) 2 (11) 0.407* 11 (55) *Expected cell count less than 5, Fisher’s Exact Test used  3 months  6 months  2000 IU (n=19)  p value  400 IU (n=20)  2000 IU (n=19)  p value  0 (0) 18 (95) 13 (68)  n/a 0.605* 0.389  0 (0) 19 (95) 18 (40)  0 (0) 18 (95) 16 (74)  n/a 1.000* 0.008*  47  3.5 3.5.1  Mean Serum 25OHD Concentrations Unadjusted Serum 25OHD Concentrations Mean (± SD) serum baseline 25OHD concentration was 65 (± 23) nmol/L in the 400 IU  group, significantly higher than the 2000 IU group of 55 (± 17) nmol/L (p = 0.010, Table 3.7). This is likely due to chance differences in the treatment arms, such as prior supplement use or UV exposure. By the 3-month study visit, the difference between groups was reversed. In ITT analysis, the 400 IU group mean serum 25OHD concentration was 72 ± 23 nmol/L, while the 2000 IU group mean serum 25OHD concentration was 85 ± 25 nmol/L (p = 0.013). At 6-months a similar difference was present, mean serum 25OHD concentration in the 400 IU group was 70 ± 22 nmol/L vs. the 2000 IU group mean of 86 ± 26 nmol/L (p=0.003). Slightly larger differences between groups were seen in the as-treated analysis (Table 3.8), although overall the numbers are very similar. Lowest 25OHD concentration measured at the 3 months follow-up visit was 31.3 nmol/L and at 6 months was 28 nmol/L, still quite close to deficiency (25 nmol/L). Reason for this low 25OHD concentration was not poor adherence as reported by the participant, but must be due to another environmental or genetic determinant.  48  Table 3.7 Mean outcome measure per group, ITT Outcome Measure Baseline (mean ± SD) Serum 25OHD (nmol/L) 400 IU, n = 40 65 ± 23 2000 IU, n = 42 55 ± 17 p-value 0.010 PCDAI 400 IU, n = 40 5.7 ± 5.7 2000 IU, n = 43 5.9 ± 6.7 p-value 0.860  3-Months (mean ± SD)  6-Months (mean ± SD)  72 ± 23 85 ± 25 0.013  70 ± 22 86 ± 26 0.003  6.7 ± 7.7 6.8 ± 7.5 0.946  8.5 ± 9.9 6.9 ± 7.9 0.405  3-Months (mean ± SD)  6-Months (mean ± SD)  65 ± 23 55 ± 17 0.010  72 ± 23 91 ± 23 0.001  70 ± 21 90 ± 26 0.001  5.7 ± 5.7 5.9 ± 6.7 0.860  7.2 ± 8.1 7.4 ± 7.8 0.905  9.6 ± 10.6 7.5 ± 8.5 0.369  Table 3.8 Mean outcome measure per group, as-treated Outcome Measure Baseline (mean ± SD) Serum 25OHD (nmol/L) 400 IU, n = 40, 34, 34 2000 IU n = 42, 35, 34 p-value PCDAI 400 IU, n = 40, 34, 32 2000 IU, n = 43, 37, 35 p-value 3.5.2  Serum 25OHD Concentrations Adjusted for Baseline Values Mean serum 25OHD concentrations were also analyzed by adjusting for baseline values.  In ITT analysis (Table 3.9) at 3-months the adjusted means of the 2000 IU group were 19 (95% CI: 10 – 29) nmol/L higher than the 400 IU group. Adjusted mean serum 25OHD concentration was 69 (95% CI: 62 – 76) nmol/L in the 400 IU group, and 88 (95% CI: 81 – 95) nmo/L in the 2000 IU group (p < 0.001). At 6-months, the difference was 24 (95% CI: 15 – 33) nmol/L. Adjusted means were 66 (95% CI: 60 – 73) nmol/L in the 400 IU group vs. 90 nmol/L (95% CI:  49  84 – 96) nmol/L in the 2000 IU group (p < 0.001). Very similar values were obtained in as treated analysis (Table 3.10).  Table 3.9 Difference in outcome measures adjusted for baseline values, ITT Outcome Measure Baseline 3-Months 6-Months Serum 25OHD (nmol/L), mean (95% CI) 400 IU*, n = 40 65 (58 - 73) 69 (62 - 76) 66 (60 - 73) 2000 IU, n = 42 55 (49 - 60) 88 (81 - 95) 90 (84 - 96) Adjusted Difference n/a 19 (10 - 29) 24 (15 - 33) p-value 0.019 < 0.001 < 0.001 BSAP (µg/L), mean (95% CI) 400 IU*, n = 40 57.4 (38.9 - 75.9) 46.5 (40.5 - 52.5) 54.8 (44.8 - 64.8) 2000 IU, n = 41 51.3 (43.2 - 59.4) 52.3 (46.4 - 58.2) 51.6 (41.7 - 61.5) Adjusted Difference n/a 5.8 (-2.6 - 14.3) -3.2 (-17.3 - 10.9) p-value 0.538 0.175 0.656 *reference group  Table 3.10 Difference in outcome measures adjusted for baseline values, as treated Outcome Measure Baseline 3-Months 6-Months Serum 25OHD (nmol/L), mean (95% CI) 400 IU*, n = 40,34,34 65 (58 - 73) 70 (63 - 77) 67 (60 - 74) 2000 IU, n = 42,35,34 55 (49 - 60) 93 (86 - 100) 93 (86 - 100) Adjusted Difference n/a 23 (14 - 33) 26 (16 - 36) p-value 0.019 < 0.001 < 0.001 BSAP (µg/L), mean (95% CI) 400 IU*, n = 40, 34, 34 57.4 (38.9 - 75.9) 46.1 (39.3 - 52.9) 56.3 (44.4 - 68.2) 2000 IU, n = 41, 34, 33 51.3 (43.2 - 59.4) 50.9 (44.1 - 57.7) 51.1 (39.0 - 63.2) Adjusted Difference n/a 4.8 (-4.9 - 14.4) - 5.3 (-22.3 - 11.8) p-value 0.538 0.326 0.541 *reference group  50  3.6 3.6.1  BSAP and Disease Activity Results Adjusted Mean Serum BSAP There was no significant difference between groups in mean BSAP concentrations when  adjusted for baseline values, as presented in Table 3.9 and Table 3.10. Additionally, mean BSAP concentrations at 6-months were compared between groups after adjustment for age and sex, in a general linear model (not shown). BSAP concentrations varied significantly with age, however sex was not a significant factor. When standardized height velocity category was added to the model, it was not a significant factor, and sex remained a non-significant factor. No significant difference in BSAP concentrations between groups was found in any model of analysis.  51  Table 3.11 Disease activity by group at each study visit, ITT Baseline 400 IU, n 2000 IU, Outcome = 40 n = 43 p value PCDAI < 10, N (%) 28 (70) 30 (70) 0.618 10 - 30, N (%) 12 (30) 12 (28) ≥ 30, N (%) 0 (0) 1 (2)  400 IU, n = 40  3 months 2000 IU, n = 43  29 (73) 11 (28) 0 (0)  27 (63) 16 (37) 0 (0)  Table 3.12 Disease activity by group at each study visit, as treated Baseline 3 months 400 IU, 2000 IU, 400 IU, 2000 IU, Outcome n = 40 n = 43 p value n = 34 n = 37 PCDAI < 10, N (%) 28 (70) 30 (70) 0.618 24 (71) 22 (60) 10 - 30, N (%) 12 (30) 12 (28) 10 (29) 15 (41) ≥ 30, N (%) 0 (0) 1 (2) 0 (0) 0 (0)  p value 0.345  p value 0.327  400 IU, n = 40  6 months 2000 IU, n = 43  26 (65) 10 (25) 4 (10)  29 (67) 14 (33) 0 (0)  6 months 400 IU, 2000 IU, n = 32 n = 35 19 (59) 9 (28) 4 (13)  23 (66) 12 (34) 0 (0)  p value 0.094  p value 0.096  52  3.6.2  Disease activity Mean PCDAI scores are presented by group at each study visit in Table 3.7. No  significant difference existed between groups at any time point, and the mean score remained <10 (remission) at each time point. Results were largely similar in ITT analysis as with the as-treated analysis Table 3.8. PCDAI scores were also analyzed by cutoff (Table 3.11) and compared between groups. At baseline, one subject in the 2000 IU group had a PCDAI of 30, 12 subjects in each treatment group had PCDAI scores 10 – 30, and the remaining were all <10. At the 3-month study visit, percentages were similar, 29 vs. 41 % of subjects had scores 10 – 30 in the 400 and 2000 IU groups respectively, and the remaining were all <10. At 6-months, 4 subjects had exceeded the 30 cutoff in the 400 IU group, compared to none in the 2000 IU group, 28% vs. 34% of the subjects had scores 10 – 30, and 59 vs. 66% of the subjects had scores <10 in the 400 and 2000 IU groups respectively. No significant difference was found between treatment groups at any timepoint. As-treated results were similar and are presented in Table 3.12.  3.7  Safety Markers The number of subjects exceeding safety cutoffs for serum calcium, serum phosphate and  urinary calcium/creatinine ratios per treatment group is reported in Table 3.13. There was no significant difference between groups at any time point. Three subjects had high serum calcium concentrations (age-specific cutoffs, see Table 2.4) at baseline, 2 at 3-months, and 1 at 6-months. Seven subjects had high serum phosphate at baseline, as did 10 subjects at each follow-up visit. Two subjects had high urinary calcium/creatinine ratio (> 0.7 mmol/mmol) at baseline, 4 at 3months (ITT), and 5 at 6-months (ITT). Number of cases exceeding safety cutoffs was generally 53  low, therefore the non-parametric Fisher’s test was used to compare the two groups. Mean biochemical measures are reported from ITT analysis in Table 3.15, and as-treated analysis in Table 3.16. There was no significant difference between groups at any study visit.  Table 3.13. Number of subjects exceeding safety marker cutoffs, ITT Safety Marker Baseline n (%) 3-Months n (%) Serum calcium > age-appropriate cutoff 400 IU, n = 40 2 (5) 2 (5) 2000 IU, n = 42 1 (2) 0 (0) p-value 0.611* 0.235* Serum phosphate > age-appropriate cutoff 400 IU, n = 39 1 (3) 7 (18) 2000 IU, n = 42 6 (14) 3 (7) p-value 0.110* 0.184* Urinary Ca/Cr > 0.7 mmol/mmol 400 IU, n = 38 2 (5) 3 (8) 2000 IU, n = 40 0 (0) 1 (2) p-value 0.234* 0.352* Urinary Ca/Cr > 0.6 mmol/mmol 400 IU, n = 38 3 (8) 4 (11) 2000 IU, n = 40 1 (3) 5 (12) p-value 0.352* 1.000* * Expected cell count less than 5, Fisher’s Exact Test used  6-Months n (%)  1 (3) 0 (0) 0.488*  4 (10) 6 (14) 0.739* 4 (11) 1 (2) 0.195* 4 (11) 3 (7) 0.708*  54  Table 3.14. Number of subjects exceeding safety marker cutoffs, as-treated Safety Marker Baseline n (%) 3-Months n (%)  6-Months n (%)  Serum calcium > age-appropriate cutoff 400 IU, n = 40, 34, 34 2 (5) 2000 IU, n = 42, 33, 34 1 (2) p-value 0.611* Serum phosphate > age-appropriate cutoff 400 IU, n = 39, 32, 34 1 (3) 2000 IU, n = 42, 31, 32 6 (14) p-value 0.110* Urinary Ca/Cr > 0.7 mmol/mmol 400 IU, n = 38, 31, 33 2 (5) 2000 IU, n = 40, 33, 31 0 (0) p-value 0.234* Urinary Ca/Cr > 0.6 mmol/mmol 400 IU, n = 38, 31, 33 3 (8) 2000 IU, n = 40, 33, 31 1 (3) p-value 0.352* * Expected cell count less than 5, Fisher’s Exact Test used  2 (6) 0 (0) 0.493*  1 (3) 0 (0) 1.000*  7 (22) 3 (10) 0.302*  4 (12) 6 (19) 0.505*  2 (7) 1 (3) 0.607*  3 (9) 1 (3) 0.614*  3 (10) 4 (12) 1.000*  3 (10) 2 (6) 1.000*  55  Table 3.15. Mean serum and urine laboratory analyses at each study visit, ITT Biochemical Measure Baseline mean ± 3-Months mean ± SD 6-Months mean ± SD SD Serum markers Hct (%) 400 IU, n = 38 2000 IU, n = 40 p-value ESR (mm/hr) 400 IU, n = 35 2000 IU, n = 36 p-value Calcium (mmol/L) 400 IU, n = 40 2000 IU, n = 42 p-value Phosphate (mmol/L) 400 IU, n = 39 2000 IU, n = 42 p-value Albumin (g/L) 400 IU, n = 40 2000 IU, n = 42 p-value Creatinine (µmol/L) 400 IU, n = 38 2000 IU, n = 42 p-value Urinary markers Phosphate (mmol/L) 400 IU, n = 39 2000 IU, n = 42 p-value Calcium (mmol/L) 400 IU, n = 39 2000 IU, n = 42 p-value Creatinine (µmol/L) 400 IU, n = 39 2000 IU, n = 42 p-value Calcium/creatinine ratio (mmol/mmol) 400 IU, n = 39 2000 IU, n = 42 p-value  0.367 ± 0.039 0.371 ± 0.041 0.638  0.370 ± 0.035 0.374 ± 0.040 0.644  0.368 ± 0.035 0.378 ± 0.045 0.246  24 ± 21 26 ± 24 0.725  22 ± 19 22 ± 23 0.970  21 ± 19 21 ± 20 0.907  2.34 ± 0.21 2.28 ± 0.24 0.281  2.29 ± 0.24 2.22 ± 0.28 0.239  2.29 ± 0.20 2.22 ± 0.25 0.165  1.46 ± 0.22 1.42 ± 0.28 0.202  1.45 ± 0.28 1.40 ± 0.22 0.370  1.40 ± 0.27 1.40 ± 0.23 0.890  41 ± 6 41 ± 6 0.665  40 ± 6 39 ± 7 0.731  41 ± 6 40 ± 7 0.472  52 ± 13 54 ± 12 0.474  53 ± 13 54 ± 12 0.854  55 ± 15 53 ± 12 0.594  19.7 ± 11.6 25.5 ± 16.9 0.143  22.7 ± 14.4 22.1 ± 12.0 0.826  23.8 ± 15.3 22.6 ± 14.2 0.723  3.6 ± 2.5 2.3 ± 1.9 0.011  3.2 ± 2.1 2.6 ± 2.1 0.257  3.3 ± 2.5 2.4 ± 2.0 0.067  13847 ± 7149 13907 ± 6744 0.749  13783 ± 6713 13678 ± 7096 0.946  14489 ± 7613 13601 ± 7340 0.594  0.28 ± 0.22 0.18 ± 0.15 0.019  0.28 ± 0.24 0.25 ± 0.24 0.499  0.31 ± 0.30 0.21 ± 0.19 0.087  56  Table 3.16. Mean serum and urine laboratory analyses at each study visit, as treated Biochemical Measure Baseline mean ± 3-Months mean ± SD 6-Months mean ± SD SD Serum markers Hct (%) 400 IU, n = 38, 36, 33 2000 IU, n = 40, 33, 35 p-value ESR (mm/hr) 400 IU, n = 35, 34, 33 2000 IU, n = 36, 31, 35 p-value Calcium (mmol/L) 400 IU, n = 40, 34, 33 2000 IU, n = 42, 33, 31 p-value Phosphate (mmol/L) 400 IU, n = 40, 34, 34 2000 IU, n = 42, 34, 32 p-value Albumin (g/L) 400 IU, n = 40, 36, 33 2000 IU, n = 42, 36, 35 p-value Creatinine (µmol/L) 400 IU, n = 38, 33, 34 2000 IU, n = 42, 34, 35 p-value Urinary markers Phosphate (mmol/L) 400 IU, n = 39, 32, 32 2000 IU, n = 42, 34, 33 p-value Calcium (mmol/L) 400 IU, n = 39, 31, 33 2000 IU, n = 42, 34, 31 p-value Creatinine (µmol/L) 400 IU, n = 39, 33, 33 2000 IU, n = 42, 34, 34 p-value Calcium/creatinine ratio (mmol/mmol) 400 IU, n = 39, 31, 33 2000 IU, n = 42, 33, 31 p-value  0.367 ± 0.039 0.371 ± 0.041 0.638  0.370 ± 0.035 0.377 ± 0.040 0.401  0.371 ± 0.034 0.380 ± 0.044 0.323  24 ± 21 26 ± 24 0.725  22 ± 19 23 ± 24 0.896  23 ± 22 22 ± 20 0.834  2.34 ± 0.21 2.28 ± 0.24 0.281  2.27 ± 0.26 2.23 ±0.28 0.490  2.28 ± 0.22 2.23 ± 0.24 0.411  1.46 ± 0.22 1.42 ± 0.28 0.202  1.45 ± 0.30 1.38 ± 0.23 0.301  1.40 ± 0.24 1.41 ± 0.29 0.825  41 ± 6 41 ± 6 0.665  39 ± 6 39 ± 7 0.942  40 ± 6 40 ± 7 0.585  52 ± 13 54 ± 12 0.474  54 ± 11 55 ± 13 0.741  56 ± 15 53 ± 13 0.394  19.7 ± 11.6 25.5 ± 16.9 0.143  24.2 ± 14.2 21.3 ± 11.9 0.377  25.5 ± 16.1 22.2 ± 14.6 0.388  3.6 ± 2.5 2.3 ± 1.9 0.011  2.6 ± 1.5 2.6 ± 2.2 0.868  3.0 ± 2.4 2.4 ± 2.0 0.312  13847 ± 7149 13907 ± 6744 0.749  13680 ± 7043 13667 ± 7228 0.994  13978 ± 7967 12861 ± 6929 0.542  0.28 ± 0.22 0.18 ± 0.15 0.019  0.26 ± 0.24 0.26 ± 0.26 0.978  0.29 ± 0.28 0.22 ± 0.19 0.260  57  Chapter 4: Discussion 4.1  Subjects achieving serum 25OHD concentration cutoffs In ITT analysis at 6-months, 88% of subjects had achieved the serum 25OHD  concentration cutoff for adequacy of 50 nmol/L, and there was no significant difference between treatment groups (400 IU: 87% vs. 2000IU: 88%, p = 0.934) (Table 3.13). The percentage of those achieving 25OHD concentrations greater than 50 nmol/L on both doses was slightly higher in as-treated analysis than ITT, 93% vs 88%. However, there were no differences in the as-treated analysis between treatment groups for those achieving 50 nmol/L (91 vs 94%, p = 0.642). Data regarding those who were 80% adherent was similar, although the sample size was smaller (n = 39). This finding supports that a daily supplement dose of 400 IU is sufficient for most children with quiescent Crohn’s disease to achieve 25OHD concentrations greater than 50 nmol/L. The only other randomized controlled trial of vitamin D supplementation in children with IBD was conducted by Pappa et al (107). After only 6-weeks of daily 2000 IU vitamin D3 supplementation Pappa et al. reported that 95% of subjects had achieved serum 25OHD concentrations > 50 nmol/L. The comparison groups used in the trial were 2000 IU D2 daily or 50,000 D2 weekly, which promoted serum 25OHD concentrations > 50 nmol/L in 75% and 95% of subjects, respectively (107). There is no other published research reporting the effect of 400 IU/d on 25OHD concentrations in children with Crohn’s disease. In the Pappa et al. trial, only 38% of children consuming 2000 IU vitamin D3/d achieved 25OHD concentrations > 75 nmol/L at 6-weeks (107). In our study, by 3-months 60% of the 2000 IU group attained 75 nmol/L, and by 6-months, 74% (ITT analysis, Table 3.13). The difference between studies may reflect a gradual accumulation in 25OHD concentrations over  58  time with prolonged supplement use, but may also be due to difference in populations, as our study only included children with inactive Crohn’s disease of any vitamin D status but consuming < 400 IU/d supplemental vitamin D per day, while Pappa et al. included all disease activity levels of only children with 25OHD concentrations that were less than 50 nmol/L at baseline. In examining the percentage of subjects achieving the 75 nmol/L cutoff within our project, there is a large difference between the treatment groups. Thirty-five percent of the 400 IU group achieved the 75 nmol/L cutoff at 6-months compared to 74% of the 2000 IU group in ITT analysis (p < 0.001). If future research were to justify a recommendation of serum 25OHD concentrations > 75 nmol/L as a target for children with Crohn’s disease, rather than 50 nmol/L, a daily dosage of 2000 IU or greater would be required, and 400 IU/d would be inadequate. There remains the question of whether a larger sample size would in fact have shown a difference in number of children achieving the 50 nmol/L cutoff between the two treatment groups. In as-treated analysis, there was a significant difference in number of children below the 50 nmol/L cutoff at 3-months, although this was not seen in ITT analysis. By 6-months this had disappeared, which could indicate that 25OHD concentrations were slower to rise in the 400 IU group.  4.2  Difference in Mean 25OHD Concentrations between Groups In ITT analysis, adjusting for baseline 25OHD concentrations, by 3-months the 2000 IU  group had a 19 (95% CI: 10 – 29) nmol/L greater adjusted mean 25OHD concentration than the 400 IU group, and by 6-months this difference had increased to 24 (95% CI: 15 – 33) nmol/L. This difference in mean 25OHD concentrations mirrors the difference between groups of children achieving 75 nmol/L concentrations, and quantifies the difference in effect between the two 59  supplement doses on 25OHD concentrations. An exploratory meta-regression of vitamin D dose to 25OHD concentration has hypothesized that each additional 100 units of vitamin D intake increases serum 25OHD concentrations by 1-2 nmol/L (109). For our study this would represent a 16-32 nmol/L difference between groups, which is almost exactly what we observed. To compare unadjusted mean serum 25OHD concentrations to the 6-week Pappa et al. trial, the final 25OHD concentrations obtained from daily 2000 IU vitamin D3 supplementation were as close as could be expected considering the different study protocol and inclusion criteria: mean serum 25OHD concentration in our study was 91 nmol/L after 3 months, or 90 nmol/L after 6 months (ITT unadjusted mean, Table 3.X), compared to 79 nmol/L after 6-weeks 2000 IU vitamin D3 in the Massachusetts study. No other trial in children with Crohn’s disease has looked at serum 25OHD concentrations under a daily vitamin D dose as low as 400 IU/d. There was a marginal increase of about 5 – 7 nmol/L in mean serum 25OHD concentrations in the 400 IU group from baseline, however without a placebo group it is difficult to rule out confounding factors. Fourteen (35%) of the 40 subjects randomized to receive 400 IU/d were consuming some supplement containing vitamin D at baseline, (mean supplemental intake = 428 IU/d), and therefore there would not be a large expected increase in serum 25OHD concentration in this subgroup. In addition, according to the previously mentioned meta-regression, we would only expect a 4-8 nmol/L increase in those who were not consuming a vitamin D supplement (109).  4.3  Baseline 25OHD Concentrations Overall, prevalence of vitamin D deficiency (25OHD < 25 nmol/L) in baseline serum  25OHD measures in our study subjects was low. Only 2 subjects were deficient and by 3-months no one was deficient. However, at baseline 33% of subjects were below the 50 nmol/L cutoff for adequacy, demonstrating a need for vitamin D supplementation in this group. This prevalence of 60  vitamin D inadequacy is similar to the prevalence of 26% reported in the healthy 12-19 year old Canadian population in the Canadian Health Measures survey (82). The overall mean 25OHD concentration at baseline was 60 (± 20.6) nmol/L. This was lower than the mean concentration reported in many (75,77,78,80,81) though not all (74,76,79) of the previous studies reporting 25OHD concentrations in children with Crohn’s disease or IBD. Generally it appears that baseline concentrations measured in our subject population are within the range of those that have been assessed in other studies. However it is difficult to draw further conclusions from this comparison as each study had slightly different inclusion criteria and different measurement techniques were used. By chance, in our study the mean baseline 25OHD concentration was higher in what became our 400 IU/d treated-group than in our 2000 IU treated-group (65 ± 23 nmol/L vs. 55 ± 17 nmol/L, p = 0.010), even though our group assignment was randomized. This difference did not obscure the effect of the different treatment doses, as the difference in serum 25OHD concentrations was completely inverted by 3-months. In addition, considering the half-life of serum 25OHD is only 15 days (68), any impact of these baseline values should have been washed out by the 6-month endpoint. Furthermore, adjustment for baseline values when comparing mean 25OHD concentrations between groups would have removed any effect of this difference. Determinants of baseline vitamin D status were consistent with previously published research with the exception of season, for which there was not a significant difference in adjusted means between groups. This may be partly due to the uneven amounts of participants recruited per season, with only 10 participants having been recruited in the summer months of June 21st to September 20th. Season was kept in the general linear model because of its well-established association with vitamin D status. Age and vitamin D intake were significant covariates. After  61  adjustment for these factors, there was a significant difference in 25OHD concentrations between white and non-white subjects of 15.3 (95% CI: 5.6 – 24.9) nmol/L.  4.4  Dietary Assessment Mean dietary vitamin D intake as assessed by the FFQ was 208 ± 134 IU/d, similar to  estimated dietary vitamin D intake in the general population. Intake data from 24-hour recalls from the Canadian Community Health Survey estimated vitamin D intake of 9 to 18 year olds to range from 212 to 320 IU/d depending on gender and household income (110). Comparing our subjects mean vitamin D intake to the RDA of 600 IU/d, there is a clear need of vitamin D supplementation in the winter months when our population would be producing minimal vitamin D endogenously. Considering that the RDA is set to promote vitamin D concentrations of 50 nmol/L in 97.5% of the population (32), it is not surprising that 87% of our subjects were able to achieve concentrations > 50 nmol/L with a 400 IU/d supplement. This supports that there is not a great difference in vitamin D absorption/metabolism between children with inactive Crohn’s disease and the general healthy population.  4.5  Safety of Treatment Regimens in this Population Generally there was no difference between groups in any of the safety measures, giving  support that both vitamin D supplement doses are safe for children with quiescent Crohn’s disease. Indeed the number of cases exceeding safety cutoffs was too low to use the chi-square test, so the non-parametric Fisher’s Exact Test was used. Serum calcium and serum phosphate were categorized as being high or normal based on age-specific cutoffs provided by the Department of Pathology and Laboratory Medicine at the Children's and Women's Health Centre of British Columbia. Urinary calcium/creatinine ratio was assessed using both the facility cutoff 62  level, 0.7 mmol/mmol, as well as 0.6 mmol/mmol, the cutoff used by Pappa et al. in their 6-week trial (Table 3.13).  Neither the Pappa et al. trial nor our own found any evidence of  hypercalcemia or hypervitaminosis D as a result of vitamin D supplementation in children with IBD (107).  4.6  PCDAI Mean PCDAI for study subjects remained below 10 at each study visit. Examining the  PCDAI categorically by disease activity showed no difference between groups in either ITT or as-treated analysis (Table 3.11, 3.12). As stated previously, scores <10 are considered to be inactive disease, scores 10 – 30 are considered to be mild disease, and above 30 to be moderate to severe disease. Interestingly, the only 4 subjects to develop moderate-severe disease activity were in the 400 IU group, however there was no statistically significant difference between groups. Due to the low number of incident cases, it is impossible to hypothesize whether this is a trend or due entirely to chance.  4.7  BSAP Three- and six- month BSAP concentrations adjusted for baseline values showed no  difference between treatment groups (ITT, p = 0.656). Although previous research has recommended that BSAP be adjusted for determinants such as sex, pubertal stage, height velocity and total bone mineral content, some of these measures, particularly the total bone mineral content, were beyond the scope of this project. However, since this is a randomized controlled trial, differences between groups should be randomly distributed across groups and remove any systematic bias in results. Furthermore, adjusting for baseline values should account for many of the between subject differences, and allow for a comparison of treatment effect between groups. 63  4.8  Limitations Recruitment lasted 15 months, but was stopped due to saturation in the recruitment  population. It was financially not feasible to prolong the recruitment beyond this period of time. Although only 83 subjects were recruited, this was sufficient to determine a significant difference between groups achieving 25OHD concentrations of 75 nmol/L, because the difference in proportion was so large. In contrast, the proportion of subjects achieving 25OHD concentrations of 50 nmol/L were so similar between groups that we would likely have needed a much greater sample size to determine a significant difference between groups. It is important to note that there were not many cases of expected outcomes being close to significant, which would have indicated a potential lack in power to determine differences between groups. If there is an effect of vitamin D supplementation on PCDAI or BSAP, it would either require a significantly larger sample size, or more precise methods of outcome measure assessment or both. Inclusion criteria proved occasionally difficult to adhere to when late information regarding supplement use, late laboratory results, or late calculation of the subjects’ height velocity as part of their PCDAI score, sometimes pushed enrolled subjects beyond the inclusion criteria cutoffs. These subjects were maintained within the study, and are the reason that some baseline characteristics means may seem to conflict slightly with the inclusion criteria. Adherence and assessment of adherence are limitations in most clinical trials, and this was no exception. Although adherence was assessed through the objective measurement of weighing pill bottles, there was still the possibility for subject dishonesty in returning unconsumed pills, as is evident from the finding of greater than 100% adherence in some subjects. This manner of adherence assessment was additionally flawed as it required subjects to remember to bring in their pill bottles to study visits, which they sometimes forgot. Seventy-eight 64  percent of subjects were self-reportedly greater than 70% adherent with the study supplements, however only 62% had greater than 80% adherence. In a systematic review of 46 studies reporting compliance to oral nutritional supplements, overall mean compliance was reported to be 78% (111). In our study, mean overall reported adherence was 82 ± 19%. In the randomized controlled trial of vitamin D supplementation by Pappa et al., compliance assessed by questionnaire was high, 79% of children were 100% compliant with the 6-week supplement regimen (107). In their adult trial, Jorgensen et al. specified that 17% of their subjects were withdrawn due to non-adherence, although the cutoff for adherence was not specified (106). The fact that reported adherence is similar in our study compared to other trials supports that 100% adherence is not a realistic target in this setting, and that the level of adherence reported here is a reasonable and believable finding, despite the limitations in assessment. Lastly, it is important to remember the specific population and conditions studied in this project, and to recognize that findings may not be generalizable beyond these circumstances. Participants were children with inactive Crohn’s who were not taking more than 400 IU/d vitamin D per day. It is possible that children with active Crohn’s disease would have reduced absorption and a smaller response in serum 25OHD concentrations. Children and families elected to enroll in this study, and therefore subjects may have been more diligent in their adherence to their vitamin regimen than an average child.  4.9  Directions for Future Research Many aspects of the potential Crohn’s disease and vitamin D interaction require further  examination. In this study we saw no effect of vitamin D supplementation on disease activity or on a bone formation biomarker. This could be because there truly does not exist an effect of  65  vitamin D intake on these outcomes, or it could be that the effect was not measurable under this study design. Increasing sample size would be necessary to determine if there is an effect of vitamin D supplementation on Crohn’s disease activity. In this study there were too few individuals who experienced even mild flaring over the study period to be able to draw a meaningful comparison between groups. Increasing sample size as well as length of follow-up would increase the likelihood of participants experiencing a flare-up. Such a study would need to be a large multicentre trial in order to accrue a sufficient number of subjects, and costs of the long duration may be high. One possibility is that the marginal benefit of vitamin D supplementation occurs when an individual transitions from low or marginal vitamin D status to adequate. Because this study did not have a true placebo, there was not a comparison group with a high prevalence of vitamin D inadequacy. Ideally, a three-armed treatment design that compares vitamin D-sufficient subjects who are supplemented to deficient subjects who are supplemented to a third group of deficient subjects who are not supplemented would offer a strong comparison of the effects of vitamin D supplementation. However it would be unethical to knowingly keep subjects vitamin D deficient if it could impair their health. This design is similar to one used in the animal studies which did show a significant impact of vitamin D supplementation on inflammation in the GI tract (104). A long-term observational study of a large cohort of children with Crohn’s disease could provide information on subjects with a wide range of vitamin D status. This would provide important evidence on whether there exists an association of vitamin D status, including individuals with inadequate vitamin D status, on Crohn’s disease activity. The sample size would have to be quite large in order to be able to control for confounding variables.  66  However, considering that children with Crohn’s disease still require adequate vitamin D for promotion of adequate bone mineral density, independent of their Crohn’s disease, there is no need to wait on further information to recommend vitamin D supplementation to children with Crohn’s disease. In addition, information from a larger and longer randomized controlled trial of similar design to the one presented here might begin to elucidate a clinical impact of vitamin D supplementation on Crohn’s disease activity or bone biomarkers. However, such trials would be expensive, and the argument could be made that there first needs to be a stronger foundation of in vitro or animal research to justify the resources required in such a trial.  67  Chapter 5: Conclusion This study was designed to determine safe and effective vitamin D supplementation recommendations for children with Crohn’s disease over a medium-length period of time. The primary objective was to determine whether a vitamin D supplement dose of 2000 IU/day would achieve a higher prevalence of serum 25OHD concentrations > 50 or 75 nmol/L compared to a control dose of 400 IU/day, in children with quiescent Crohn’s disease. The cutoff of 50 nmol/L was selected to be consistent with current recommendations of adequate vitamin D status put forward by the IOM and Health Canada for the healthy population. After 6-months of supplementation, it was determined that there was no significant difference in percentage of children with serum 25OHD concentrations above 50 nmol/L between the two supplement doses (87% vs. 88%, p = 0.934). Significantly less subjects in the 400 IU/d group achieved serum 25OHD concentrations of 75 nmol/L than in the 2000 IU/d group (35% vs. 79%, p <0.001). Both regimens were found to be equally safe, with very low numbers of participants exceeding safety measure cutoffs levels, and with no significant difference between groups. Vitamin D supplementation was not found to affect secondary objective outcome measures of serum BSAP concentration, or the PCDAI score. There is a possibility that study design limitations such as duration or sample size could have impaired detection of an effect on these secondary objectives. Additionally, since almost all subjects achieved adequate vitamin D status, there was not a comparison group of subjects of insufficient vitamin D status, rather it was a comparison between subjects with adequate status and a higher adequate status. Currently there is insufficient evidence to support a target 25OHD concentration of 75 nmol/L in this population, therefore by default, recommendations should follow those established for the healthy population, 50 nmol/L. In this light a daily supplement dose of 400 IU appears to  68  be equally effective and safe as a daily supplement dose of 2000 IU in promoting adequate vitamin D status in children with quiescent Crohn’s disease. Data presented here can inform healthcare workers and families of appropriate vitamin D supplementation recommendations for children with inactive Crohn’s disease. Achievement of adequate vitamin D status will help promote adequate bone mineral accrual as well as sustain vitamin D’s many other roles throughout the body.  69  References 1.  Abraham C, Cho JH. MECHANISMS OF DISEASE Inflammatory Bowel Disease. N. Engl. J. Med. 2009 Nov 19;361(21):2066–78.  2.  Khor B, Gardet A, Xavier RJ. Genetics and pathogenesis of inflammatory bowel disease. Nature. 2011 Jun 16;474(7351):307–17.  3.  Spehlmann ME, Begun AZ, Burghardt J, Lepage P, Raedler A, Schreiber S. Epidemiology of inflammatory bowel disease in a German twin cohort: Results of a nationwide study. Inflammatory Bowel Diseases. 2008;14(7):968–76.  4.  Loftus Jr EV. Clinical epidemiology of inflammatory bowel disease: incidence, prevalence, and environmental influences. Gastroenterology. 2004 May;126(6):1504–17.  5.  Calkins BM. A meta-analysis of the role of smoking in inflammatory bowel disease. Digestive Diseases and Sciences. 1989;34(12):1841–54.  6.  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Clinical Nutrition. 2012 Jun;31(3):293–312.  78  Appendices Appendix A Study Documents A.1 Letter of First Contact  Department of Pediatrics [Contact Information]  Patient Name Address BC Dear Person (Insert Name) We are sending you this letter because you have Crohn’s Disease. We obtained your name from the database held by the Gastroenterology Clinic at BCCH. The goal of this letter is to let you know about a study that we will be conducting that involves children who have Crohn’s Disease and are between the ages of 8-18 years of age. The purpose of this study is to determine if the current recommended dose of vitamin D is enough to maintain optimal levels of blood vitamin D. We ultimately hope to determine if taking Vitamin D will help keep Crohn’s Disease under control. The study involves coming to British Columbia Children’s Hospital three times over a 6-month period. During this time you will be asked to take a Vitamin D supplement and have blood and urine collected at each visit. Please find enclosed a copy of the subject “informed consent” form. This document will provide you with information regarding the study. Should you be interested in participating in this study, please call Dr. Tim Green at [contact number]. Alternatively, if you have previously said we cab contact you about research studies we will contact you by phone within 1-2 weeks of you receiving this letter to see whether you would be interested in participating in the study. You are under no obligation to participate, and your decision will not affect your care in the Gastroenterology Clinic. Yours sincerely, Dr. Kevan Jacobson, MB BCh, FRCPC Clinical Associate Professor Faculty of Medicine Pediatrics University of British Columbia Gastroenterologist British Columbia’s Children’s Hospital Phone: [contact number]  Dr Tim Green, PhD Associate Professor Food, Nutrition, and Health University of British Columbia Phone: [contact number]  79  A.2  Study Consent Form  Department of Pediatrics [Contact Information]  Vitamin D in Pediatric Crohn’s Disease Informed Consent Form Principal Investigator  Dr. Tim Green, PhD Food, Nutrition, and Health, University of British Columbia  Co-investigator  Dr. Kevan Jacobson, MD Pediatrics, University of British Columbia  Sponsor  Food, Nutrition, and Health Vitamin Research Fund Faculty of Land and Food Systems  Site  Children’s and Women’s Health Centre of British Columbia  Contact Numbers  If you have any questions regarding this study, you may contact Dr Tim Green at [contact number] or Dr. Kevan Jacobson at [contact number]. If you have an emergency related to your Crohn’s disease or please contact the paging service at [contact number] and request to have the gastroenterologist on call paged. This service is available 24 hours a day, 7 days a week for emergency. If you have any concerns about your rights as a research subject and/or your experiences while participating in this study, contact the Research Subject Information Line in the University of British Columbia Office of Research Services by email at [email] or by phone at [contact number].  80  Introduction You (“you” refers to you, your child, or your ward throughout this consent) are being invited to take part in this research study because you are a child or adolescent with (pediatric) Crohn’s disease. Most children’s vitamin supplements contain 400 IU of vitamin D. This may not be enough for pediatric patients with Crohn’s Disease to attain high enough blood vitamin D concentrations for health. The purpose of this study is to determine if a higher dose of vitamin D (2000 IU) in patients with pediatric Crohn’s disease is better than 400 IU. We would also like to determine if the higher dose of vitamin D is better at preventing a flare up of Crohn’s disease than a lower dose. This study is being conducted at Children’s and Women’s Health Centre of British Columbia and McMaster Children's Hospital. About 100 pediatric Crohn’s Disease patients will participate in the study. Subjects will be randomized (like flipping a coin) to receive daily 400 IU or 2000 IU of vitamin D for six months. Participation Your participation is entirely voluntary, so it is up to you to decide whether or not to take part in this study. Before you decide, it is important for you to understand what the research involves. This consent form will tell you about the study, why the research is being done, what will happen to you during the study and the possible benefits, risks and discomforts. If you wish to take part in the study, you will be asked to sign this form. If you do decide to take part in this study, you are still free to withdraw at any time and without giving any reasons for your decision. If you do not wish to take part, you do not have to provide any reason for your decision not to participate nor will you lose the benefit of any medical care to which you are entitled or are presently receiving. Please take time to read the following information carefully and to discuss it with your family, friends, and doctor before you decide. Who is conducting the study? This study is being funded, in part, through in kind support provided by the Food, Nutrition, and Health Vitamin Research Fund (Faculty of Land and Food Systems). Researchers in Human Nutrition and Pediatric Gastroenterology at the University of British Columbia are conducting this study. Background Studies suggest that attaining an optimal blood level of vitamin D may be important for the prevention and treatment of Crohn’s Disease. Our long-term goal is to conduct a large multicentre randomized control trial to determine if vitamin D supplementation is effective in helping children with Crohn’s Disease patients in remission (no disease activity). At present we do not have enough information to know the best dose of Vitamin D for children with Crohn’s Disease. There is a lack of agreement on what optimal blood vitamin D levels are in a pediatric population. However, the Canadian Pediatric Society recommends suggests that blood vitamin D levels for children be above 75 nmol/L. We are conducting a six month randomized study designed trial with two doses of vitamin D (400 IU, and 2000 IU) in pediatric patients with Crohn’s Disease who are in remission.  81  Who Can Participate? Pediatric Crohn’s disease patients who are: • 8-18 years old • currently in remission • curently taking 400 IU or less vitamin D • not taking steroids Who Should not Participate in This Study? Pediatric Crohn’s disease patients who: • have active disease • are taking more than 400 IU day supplemental vitamin D • are taking corticosteroids STUDY PROCEDURES Study Procedures If you agree to participate, you will be asked to attend three appointments at the Children’s and Women’s Health Centre of British Columbia. Each of the appointment will last approximately 1 hour. The first and third appointments can be done as part of your regularly scheduled clinic visit. Visit 1 (Time 1 hour) • You will be asked questions about the severity of your disease to determine your Pediatric Crohn’s Disease Activity Index Score. The questionnaire consists of a patient recall of symptoms, laboratory values, and a clinical examination by your doctor. • You will be weighed and your height recorded. • You will allow a nurse or other certified person to take a small blood sample of 5 ml ( 1 teaspoon). The blood will be used to measure vitamin D and other related factors. • You will provide a small urine sample, which we will measure, minerals in such as calcium. • You will be asked to complete a questionnaire, which includes questions on vitamin and mineral supplement use. • You will be asked to complete a food questionnaire to determine your intake of calcium and vitamin D over the previous month. • You will stop taking any vitamin and mineral supplements you are currently taking. • You will be randomized (like flipping a coin) to take supplements that contain one of two amounts of vitamin D. • You will be asked to take the supplement that you are assigned for 6 months. Visit 2 and 3 (Time - 30 minutes each) • You will be asked questions about the severity of your disease to determine your Pediatric Crohn’s Disease Activity Index Score. The questionnaire consists of a patient recall of symptoms, laboratory values, and a clinical examination by your doctor. • You will be weighed and your height recorded • You will allow a nurse or other certified person to take a small blood sample of 5 ml (1 teaspoons). The blood will be used to measure vitamin D and other related factors. • You will provide a small urine sample 82  • •  Other You will allow the investigators to access your health records that are maintained on a database by the Pediatric Gastroenterology Service solely to collect relevant information on your previous medical background. This will include age of onset, type of Crohn’s Disease (small versus large bowel), number of prior flare-ups, prior medications and supplement use.  Risks It is felt that there are little known risks participating in this study. There is no placebo and all participants will receive at least the amount of vitamin D commonly present in most multivitamins. Very high intakes of Vitamin D (much higher than amounts used in this study) may cause an increase in blood calcium levels, which can cause mental confusion and heart arrhythmias. We will measure blood calcium levels at each visit to make sure it is not high. We will ask you to stop using any other vitamin and mineral supplements you are currently taking that contain vitamin D. However, we will provide you with a chewable vitamin and mineral supplement that does not contain vitamin D to take instead if you want to. Taking a blood sample is felt to have very low risks. The needles used to take blood might be uncomfortable and you might get minor bruising or, very rarely, an infection at the site of the needle poke. Taking a blood sample occasionally causes one to feel lightheaded, faint and/or dizzy. The blood samples provided by you/you will not be used for any purposes other than this study. We can put a cream on your arm where the blood is drawn to help with the discomfort. Benefits If you agree to take part in this study, there may or may not be a direct benefit to you. We will provide you with free supplements. Also, we will monitor your vitamin D blood levels. If we find the best amount of vitamin D, this information will be used to develop better vitamin supplements and treatments for use in Crohn’s Disease. You will be given gift certificates in the amount of $50 for taking part in this study. In addition, we will provide you with parking voucher or a transit pass for each visit. No receipts will be necessary. What are the alternatives to the study treatment? If you choose not to take part in this study you can stay on your current regimen of vitamins and minerals. The vitamins used in this study will not replace any of the current medications you are taking. Your doctor can discuss these options and their risks and benefits with you if you wish before deciding whether or not to take part in this study. You do not have to participate in this study to receive treatment for your Crohn’s disease. Confidentiality In this study you will be identified by a study code and any identifying information will be kept behind locked doors. No records, which identify you by name, initials or date of birth will be allowed to leave the Investigators' offices. Your confidentiality will be respected. No information that discloses your identity will be released or published without your specific consent to the disclosure. However, research records and medical records identifying you may be inspected in the presence of the Investigator or his or her designate by representatives of Health Canada, and the University of British Columbia Research Ethics Boards for the purpose of monitoring the research. However, no records which identify you by name or initials will be allowed to leave the Investigators' offices. 83  Your rights to privacy are protected and guaranteed by the “Freedom of Information and Protection of Privacy Act of British Columbia”. This act lays down the safeguards respecting your privacy, and also gives you the right of access to the information about you that has been provided to the study, and if needed, you have the chance to correct any errors in this information. Further details about this act are available on request. Consent This study has been explained to you and you have been given the chance to ask questions about taking part in this study. If you have questions you can ask Dr. Tim Green at [contact number] or Dr. Kevan Jacobson at [contact number]. Participation and Withdrawal from this Study Taking part in this study is voluntary. You may choose not to take part or may leave the study at any time and do not have to give a reason for your decision. If you decide not to take part or decide to leave the study, you will continue to receive the best medical care available. You will be given a copy of this signed and dated consent form. Compensation for Injury Signing this consent form in no way limits your legal rights against the sponsor, investigators, or anyone else. Your signature on the consent form means the following: • The study has been explained to you and all of your questions have been answered. • You understand what the study requires and the risks of the study; and • You agree to take part in this study.  84  Consent Form • • • • •  • • • •  I have read and understood the subject information and consent form. I have had sufficient time to consider the information provided and to ask for advice. I have had the opportunity to ask questions and have had satisfactory response to my questions. I understand that all of the information collected will be kept confidential and that the result will only be used for scientific objectives. I understand that participation in this study is voluntary and that I am completely free to refuse to participate or to withdraw from this study at any time without changing in any way the quality of care that I receive. I understand that I am not waiving any of my legal rights or my child’s as a result of signing this consent form. I have read this form and I freely consent to participate in this study. I have been told that I will receive a dated and signed copy of this form The parents/legal guardian and the investigator are satisfied that the information contained in this consent form was explained to the child to the extent that he/she is able to understand it, that all questions have been answered, and that the child assents to participating in the research.  _________________________ Printed Name of Parent/ Legal Guardian  ______________________________ ____________ Signature of Parent /Legal Guardian Date  _________________________ Printed Name of Person explaining consent  ______________________________ ____________ Signature of Person explaining consent Date  _________________________ Printed Name of Principal Investigator  ______________________________ ____________ Signature of Principal Investigator Date  SUBJECT’S ASSENT TO PARTICIPATE IN RESEARCH I have had the chance to read this form, to ask questions about this study, and to talk about it with my parents/guardians. I have had answers to all my questions. I understand that I may quit this study at any time and that I will still receive good care. I will be given a copy of this form. I agree to participate in this study. _________________________ Name of subject (print)  _______________________ Signature of Subject  ______________ Date  85  A.3  Study Assent Form  Department of Pediatrics [Contact Information]  	
   	
   SUBJECT	
  ASSENT	
  FORM	
   Children	
  aged	
  8-­‐13	
  years	
   	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
   Title	
  of	
  Study:	
  Vitamin D in Pediatric Crohn’s Disease	
   	
   Principal	
  Investigator:	
  Dr.	
  Tim	
  Green,	
  PhD	
   	
   Co	
  Investigator:	
  Dr	
  Kevan	
  Jacobson,	
  MD	
   _____________________________________________________________________________	
   I	
  am	
  being	
  invited	
  to	
  be	
  part	
  of	
  a	
  research	
  study.	
  A	
  research	
  study	
  tries	
  to	
  find	
  better	
  treatments	
   to	
  help	
  children	
  like	
  me.	
  It	
  is	
  up	
  to	
  me	
  if	
  I	
  want	
  to	
  be	
  in	
  this	
  study.	
  No	
  one	
  will	
  make	
  me	
  be	
  part	
   of	
  this	
  study.	
  Even	
  if	
  I	
  agree	
  now	
  to	
  be	
  part	
  of	
  the	
  study,	
  I	
  can	
  change	
  my	
  mind	
  later.	
  No	
  one	
  will	
   be	
  upset	
  with	
  me	
  if	
  I	
  choose	
  not	
  to	
  be	
  part	
  of	
  this	
  study.	
   	
   The	
  following	
  pages	
  will	
  tell	
  me	
  about	
  the	
  study.	
  They	
  will	
  tell	
  me	
  what	
  will	
  happen	
  if	
  I	
  decide	
  to	
   take	
   part.	
   When	
   I	
   read	
   these	
   pages	
   it	
   is	
   important	
   that	
   I	
   understand	
   everything.	
   If	
   something	
   isn’t	
  clear	
  to	
  me,	
  I	
  can	
  ask	
  the	
  researcher	
  or	
  doctor	
  to	
  explain.	
   	
   Why	
  are	
  we	
  doing	
  this	
  study?	
   I	
  am	
  invited	
  to	
  take	
  part	
  in	
  a	
  research	
  study	
  because	
  I	
  am	
  between	
  8	
  and	
  18	
  years	
  old	
  and	
  I	
  have	
   Crohn’s	
  Disease	
  (CD).	
  CD	
  is	
  soreness	
  in	
  the	
  tummy	
  that	
  can	
  cause	
  stomach	
  pain	
  and	
  diarrhea	
   (pooping	
  many	
  times	
  a	
  day).	
  This	
  research	
  is	
  looking	
  at	
  how	
  much	
  Vitamin	
  D	
  I	
  need	
  to	
  take	
  as	
  a	
   pill	
  to	
  raise	
  my	
  blood	
  vitamin	
  D	
  to	
  the	
  best	
  level.	
  We	
  are	
  comparing	
  the	
  effects	
  of	
  two	
  amounts	
   of	
  vitamin	
  D	
  on	
  blood	
  vitamin	
  D	
  levels	
  over	
  6	
  months.	
  The	
  research	
  team	
  believes	
  that	
  Vitamin	
   D	
  may	
  play	
  an	
  important	
  part	
  in	
  possibly	
  preventing	
  or	
  treating	
  IBD.	
   	
  	
    86  Who	
  is	
  doing	
  this	
  study?	
   Dr.	
  Green	
  a	
  researcher	
  in	
  nutrition	
  at	
  the	
  University	
  of	
  British	
  Columbia	
  and	
  Dr.	
  Jacobson	
  from	
   British	
  Columbia	
  Children’s	
  Hospital	
  (BCCH)	
  will	
  be	
  doing	
  this	
  study.	
  They	
  will	
  answer	
  any	
   questions	
  I	
  have	
  about	
  the	
  study.	
  I	
  can	
  also	
  call	
  them	
  if	
  I	
  am	
  having	
  any	
  problems	
  or	
  if	
  there	
  is	
   an	
  emergency	
  at	
  [contact	
  number]	
  and	
  ask	
  to	
  have	
  the	
  Gastroenterology	
  doctor	
  on	
  call	
  to	
  be	
   paged.	
   What	
  will	
  happen	
  during	
  the	
  study?	
   If	
  I	
  agree	
  to	
  take	
  part	
  in	
  this	
  study,	
  I	
  will	
  need	
  to	
  come	
  to	
  BCCH	
  to	
  see	
  the	
  researchers	
  three	
   times	
  in	
  six	
  months.	
  Two	
  of	
  the	
  visits	
  can	
  be	
  part	
  of	
  my	
  regular	
  visits	
  to	
  BCCH.	
  Each	
  visit	
  will	
  last	
   between	
  30	
  minutes	
  and	
  1	
  hour.	
  	
   	
   At	
  the	
  first	
  study	
  visit,	
  my	
  family	
  and	
  I	
  will	
  be	
   asked	
  to	
  complete	
  a	
  form	
  about	
  the	
  food	
   that	
  I	
  have	
  been	
  eating.	
   	
   	
   	
  	
    	
    At	
  the	
  first	
  visit	
  I	
  will	
  also	
  be	
  asked	
  to	
   complete	
  a	
  form	
  about	
  vitamin	
  supplements	
  I	
   take.	
  	
   	
   	
   At the first visit I will be randomized (like flipping a coin) to pills containing one of two amounts of vitamin D. I will take one pill every day for six months. 	
    	
    	
   At	
  each	
  study	
  visit	
  I	
  will	
  have	
  about	
  5	
  ml	
  (1	
  teaspoon)	
  of	
  blood	
   taken	
  to	
  check	
  how	
  much	
  Vitamin	
  D	
  I	
  have	
  in	
  my	
  body	
  and	
  I	
  will	
   provide	
  a	
  small	
  amount	
  of	
  urine.	
  	
   	
   	
    At	
  each	
  visit	
  I	
  will	
  be	
  asked	
  about	
  any	
  problems	
  I	
  am	
  having	
  such	
   as	
  a	
  tummy	
  ache	
  or	
  diarrhea	
   	
   87  100	
  other	
  young	
  people	
  with	
  CD	
  will	
  also	
  be	
  in	
  the	
  study	
   	
   	
   	
   Are	
  there	
  good	
  things	
  about	
  the	
  study?	
   A	
  good	
  thing	
  about	
  being	
  in	
  the	
  study	
  is	
  that	
  the	
  information	
  that	
  I	
  give	
  the	
  study	
  team	
  may	
   help	
  me	
  and	
  other	
  people	
  with	
  CD	
  in	
  the	
  future.	
   	
   Are	
  there	
  bad	
  things	
  about	
  the	
  study?	
   When	
  blood	
  is	
  taken	
  from	
  my	
  arm	
  it	
  may	
  make	
  me	
  uncomfortable	
  or	
  may	
  even	
  hurt	
  a	
  little.	
  	
   Sometimes	
  I	
  may	
  even	
  get	
  a	
  little	
  bruising	
  or	
  bleeding	
  where	
  the	
  needle	
  is	
  inserted	
  into	
  my	
  skin.	
   A	
  cream	
  can	
  be	
  put	
  on	
  my	
  skin	
  to	
  make	
  it	
  numb	
  before	
  the	
  needle	
  goes	
  in.	
  	
  	
   	
   Are	
  there	
  any	
  other	
  treatments	
  for	
  me?	
   I	
  do	
  not	
  have	
  to	
  be	
  in	
  this	
  study	
  to	
  be	
  treated	
  for	
  my	
  Crohn’s	
  Disease.	
  Other	
  treatments	
  are	
   available	
  and	
  I	
  can	
  ask	
  my	
  doctor	
  about	
  these.	
   	
   Who	
  will	
  know	
  about	
  what	
  I	
  did	
  in	
  the	
  study?	
   Only	
  my	
  doctors	
  and	
  people	
  who	
  are	
  involved	
  in	
  the	
  study	
  will	
  know	
  I	
  am	
  in	
  it.	
  When	
  the	
  study	
   is	
  finished,	
  the	
  doctors	
  will	
  write	
  a	
  report	
  about	
  what	
  was	
  learned.	
  This	
  report	
  will	
  not	
  say	
  my	
   name	
  or	
  that	
  I	
  was	
  in	
  the	
  study.	
  My	
  parents	
  or	
  guardian	
  and	
  I	
  do	
  not	
  have	
  to	
  tell	
  anyone	
  I	
  am	
  in	
   the	
  study	
  if	
  we	
  don’t	
  want	
  to.	
   If	
   Dr.	
   Green	
   or	
   Dr.	
   Jacobson	
   feels	
   my	
   health	
   may	
   be	
   in	
   danger,	
   they	
   may	
   have	
   to	
   report	
   the	
   results	
  to	
  my	
  other	
  doctor.	
   	
   When	
  Can	
  I	
  decide	
  if	
  I	
  want	
  to	
  be	
  in	
  the	
  study?	
   I	
  have	
  as	
  much	
  time	
  as	
  I	
  want	
  to	
  decide	
  to	
  be	
  part	
  of	
  this	
  study.	
  	
  Nobody	
  will	
  be	
  angry	
  or	
  upset	
  if	
   I	
  do	
  not	
  want	
  to	
  be	
  in	
  the	
  study.	
  	
  The	
  study	
  doctors	
  are	
  talking	
  to	
  my	
  parent	
  or	
  legal	
  guardians	
   about	
  the	
  study	
  and	
  I	
  should	
  talk	
  to	
  them	
  about	
  it	
  too.	
    88  	
   	
   ASSENT	
  STATEMENT	
   	
   If	
  I	
  put	
  my	
  name	
  at	
  the	
  end	
  of	
  this	
  form,	
  it	
  means	
  that	
  I	
  agree	
  to	
  be	
  in	
  the	
  study.	
  	
  I	
  will	
  receive	
   a	
  signed	
  and	
  dated	
  copy	
  of	
  this	
  assent	
  form	
  for	
  my	
  records.	
   	
   	
   I	
  assent	
  to	
  participate	
  in	
  this	
  study.	
   	
   Child’s	
  Name:	
  __________________________________	
   	
   Child’s Signature: __________________________  Date:________________________  89  Appendix B Pediatric Crohn’s Disease Activity Index  Pediatric Crohn’s Disease and Vitamin D supplementation PCDAI Checklist History (Recall, 1 week) Abdominal pain: None Mild – Brief, does not interfere with activities Mod/severe – daily, longer lasting, affects activities, nocturnal  _____(0) _____(5) _____(10)  Stools: (per day) 0-1 liquid stools, no blood Up to 2 semi-formed with small blood, or 2-5 liquid Gross bleeding, or ≥ liquid, or nocturnal diarrhea  _____(0) _____(5) _____(10)  Patient Functioning, General Well-Being (Recall, 1 week) No limitation of activities, well Occasional difficulty in maintaining age appropriate activities, below par Frequent limitation of activity, very poor  _____(0) _____(5) _____(10)  Laboratory HCT (%) <10 yrs: 11-19F:  ESR (mm/hr)  >33 _____(0) 28-32 _____(2.5)  11-14M:  >34 _____(0) 29-33 _____(2.5) <29 _____(5)  15-19M:  <20 20-50 >50  ≥35 ____(0) 30-34 ____(2.5) ≥37 ____(0) 32-36 ____(2.5) <32 ____(0)  _____(0) _____(2.5) _____(5)  Albumin (g/dL) >35 _____(0) 31-34 _____(5) <30 _____(10)  90  EXAMINATION Weight Weight gain or voluntary weight stable/loss Involuntary weight stable, weight loss 1-9% Weight loss > 10%  _____(0) _____(5) _____(10)  Height At Diagnosis: <1 channel decrease >1, <2 channel decrease >2 channel decrease or Follow-up:* Height velocity >-1SD Height velocity <-1SD, >-2SD Height velocity <-2SD  _____(0) _____(5) _____(10) _____(0) _____(5) _____(10)  Abdomen No tenderness, no mass Tenderness, or mass without tenderness Tenderness, involuntary guarding, definite mass Perirectal disease None, asymptomatic tags 1-2 indolent fistula, scant drainage, no tenderness Active fistula, drainage, tenderness, or abscess  _____(0) _____(5) _____(10) _____(0) _____(5) _____(10)  Extra-intestinal Manifestation (Fever >38.5 for 3 days over past week, definite arthritis, uveitis, E.nodosum, P. gangrenosum) None _____(0) One _____(5) > Two _____(10) Total Score  _______________________________  91  Appendix C Food Frequency Questionnaire  92  93  94  

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