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Vitamin D intake and vitamin D status in 5 - 6 year old children in Vancouver Rasmussen, Betina Feldfoss 2013

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VITAMIN D INTAKE AND VITAMIN D STATUS IN 5 - 6 YEAR OLD CHILDREN IN VANCOUVER  by  Betina Feldfoss Rasmussen  B.Sc., The Metropolitan University College, Copenhagen Denmark, 2011  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE AND POSTDOCTORIAL STUDIES  (Human Nutrition)  THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)     October 2013  ? Betina Feldfoss Rasmussen, 2013   ii Abstract Vitamin D is important in maintaining bone health and has recently been proposed to have additional roles in the immune system and brain development. The estimated average requirement (EAR) and recommended dietary allowance (RDA) for vitamin D established by the Institute of Medicine (IOM) in 2011 is 10?g/day and 15?g/day, respectively. When this study was initiated, little information was available on whether vitamin D intakes below the recommendations in young children result in biochemical evidence of vitamin insufficiency or deficiency. Therefore the aims in this thesis were; to estimate vitamin D intakes in children, and the contribution of natural and fortified foods, and supplements; to determine the proportion of children consuming vitamin D below and above the intake recommendations; to use biochemical measures of plasma 25(OH)D to determine the proportion of vitamin D sufficient, insufficient and deficient children; and to estimate the importance of vitamin D intake and season to the children?s plasma 25(OH)D. This was a cross-sectional design with 200 children from Vancouver BC, aged 5.75 years. Vitamin D intakes were estimated using a food frequency questionnaire and 24 hr dietary recalls. Plasma 25 (OH)D was determined by HPLC-tandem mass spectrometry. The median vitamin D intake from foods was below the EAR and RDA. The children obtained 85.9% of their dietary vitamin D from supplements and fortified foods and 14.1% from natural food sources. Total median vitamin D intakes in children given or not given supplements was 13.0 (9.0) ?g/day and 4.8 (3.7) ?g/day, respectively, P< 0.001. Using the FFQ, 51% and 76% of the children did not meet the EAR and RDA for vitamin D, respectively. However, only 4.7% and 19.0% had a plasma 25 (OH)D below 40 nmol/L or 50 nmol/L, respectively. Unexpectedly, only 12.5% of the children who did not meet the EAR during winter months had a plasma 25 (OH)D below 40 nmol/L. The results in this thesis suggest that children depend on supplements and fortified foods to achieve the current vitamin D intake   iii recommendations. However, despite apparent low vitamin D intakes, few children show biochemical evidence of vitamin D insufficiency, even during winter months.                                  iv Preface This thesis presents work conducted by myself, Betina Rasmussen, under the supervision of Dr. Sheila Innis, with ongoing guidance from Dr. David Kitts and Dr. Tim Green at the University of British Columbia.  This project was part of a larger research study, examining essential fatty acid requirements to support optimal childhood development and health.  The study protocol was designed by principal investigator Dr. Sheila Innis and funded primarily by her funding from the Canadian Institutes of Health Research and Freedom to Discover awards. The project involves team members with complementary expertise, including Kelly Richardson, with graduate training in child developmental assessments, other graduate students, including Kelly Mulder and Brain Wu, and expert laboratory technicians including Roger A Dyer and Janette D King.  My roles in this project included conducting parent interviews, including dietary intake data collection using 24 hr recalls and Food Frequency Questionnaires, and collection of dietary data using 24 hour recalls by telephone. I was responsible with Kelly Mulder for entry of all of the dietary data into the nutrient bases, and I estimated the vitamin D intakes for all diet records. I assisted in sample preparation for the analysis of plasma 25(OH)D analysis by LC-MS/MS, which was done  by Roger Dyer. I was responsible for summarizing all of the data relevant to this thesis, conducted all of the statistical analysis in this thesis and wrote the thesis with supervision from Dr. Sheila Innis. Sections of this thesis will be submitted for publication as a manuscript to academic journals. The University of British Columbia Children and Women?s Clinical Research Ethics Board approved this study protocol (identifiers: H09-02921 and H09-01633).  I was supported from my graduate training by Statens Uddannelsesst?tte (SU) from the Danish government and from grant funds held by Dr Innis.    v Table of contents Abstract ......................................................................................................................................... ii Preface ........................................................................................................................................... iv Table of contents ............................................................................................................................ v List of tables ............................................................................................................................... viii List of figures.................................................................................................................................. x List of abbreviations ..................................................................................................................... xi Acknowledgements ..................................................................................................................... xii Chapter 1: Literature review .................................................................................................. 1 1.1 Introduction ............................................................................................................................. 1 1.2 Vitamin D sources ................................................................................................................... 2 1.2.1 Diet, supplements, fortification and cutaneous synthesis ................................................... 2 1.3 Vitamin D metabolism ............................................................................................................ 7 1.3.1 Absorption and transport ..................................................................................................... 7 1.3.2 Hydroxylation of vitamin D in the liver and kidney ........................................................... 7 1.3.3 Physiological roles of vitamin D ......................................................................................... 8 1.3.3.1 Calcium and phosphate homeostasis and bone health ................................................ 9 1.3.3.2 Emerging roles of vitamin D .................................................................................... 11 1.4 Consequences of low vitamin D status .................................................................................. 12 1.5 Factors affecting vitamin D status ......................................................................................... 12 1.5.1 Individual factors affecting vitamin D status .................................................................... 12 1.5.2 Environmental and lifestyle factors affecting vitamin D status ........................................ 13 1.6 Assessment of vitamin D status ............................................................................................. 14 1.6.1 Vitamin D sufficiency, insufficiency and deficiency ........................................................ 14   vi 1.7 Vitamin D intake recommendations ...................................................................................... 16 1.8 Current knowledge of vitamin D intake and status in children ............................................. 17 1.8.1 Canadian community health measures survey cycle 2.2 - 2004 ....................................... 17 1.8.2 Canadian health measures survey 2007 - 2009 and 2009 - 2011 ...................................... 18 1.8.3 USA ? National health and nutrition examination survey ................................................ 19 1.8.4 Research reports ................................................................................................................ 20 1.8.4.1 Canada ...................................................................................................................... 20 1.8.4.2 U.S.A ........................................................................................................................ 22 1.8.5 Summary ........................................................................................................................... 24 Chapter 2: Study .................................................................................................................... 30 2.1 Purpose .................................................................................................................................. 30 2.2 Objectives .............................................................................................................................. 31 2.3 Methods ................................................................................................................................. 31 2.3.1 Design and setting ............................................................................................................. 31 2.3.2 Inclusion and exclusion criteria and recruitment .............................................................. 32 2.3.3 Demographic characteristics ............................................................................................. 32 2.3.4 Dietary assessments and collection of information on supplement use ............................ 33 2.3.5 Analysis of dietary intakes ................................................................................................ 34 2.3.6 Anthropometrics ................................................................................................................ 35 2.3.7 Blood collection, preparation and analysis of 25(OH)D ................................................... 35 2.3.7.1 LC-MS/MS ............................................................................................................... 35 2.3.8 Statistical analysis ............................................................................................................. 37 Chapter 3: Results .................................................................................................................. 39 3.1 Subject characteristics ........................................................................................................... 40 3.2 Vitamin D intake ................................................................................................................... 44 3.2.1 Vitamin D intakes from foods including natural sources and fortified foods ................... 48 3.2.2 Vitamin D intakes from supplements ................................................................................ 49   vii 3.2.3 Vitamin D intake from foods and supplements ................................................................. 50 3.2.4 Vitamin D intakes compared to the EAR and RDA for children ...................................... 52 3.3 Plasma 25 (OH)D .................................................................................................................. 58 3.3.1 Plasma 25 (OH)D and vitamin D intake of children during different seasons .................. 59 3.3.2 Plasma 25 (OH)D of children given and not given supplemental vitamin D during different seasons ............................................................................................................................. 61 Chapter 4: Discussion ............................................................................................................ 63 4.1 Vitamin D intake ................................................................................................................... 63 4.1.1 Vitamin D sources ............................................................................................................. 64 4.1.2 Vitamin D intake from supplements ................................................................................. 65 4.1.2.1 Total vitamin D intake .............................................................................................. 67 4.1.3 Vitamin D intake compared to the EAR and RDA ........................................................... 68 4.1.4 Vitamin D intakes assessed using FFQ and 24 hr recalls ................................................. 69 4.2 Plasma 25 (OH)D .................................................................................................................. 71 4.2.1 Season ............................................................................................................................... 72 4.2.2 Relationship of vitamin D intake to vitamin D status ....................................................... 73 4.3 Strengths and limitations ....................................................................................................... 76 4.4 Future directions .................................................................................................................... 78 Chapter 5: Conclusion ........................................................................................................... 79 References ..................................................................................................................................... 80 Appendices ................................................................................................................................... 87 Appendix A Informed consent form ................................................................................................... 87 Appendix B Socio-demographic questionnaire .................................................................................. 95 Appendix C Food frequency questionnaire ........................................................................................ 97     viii List of tables Table 1.1  Vitamin D content in foods .............................................................................. 4 Table 1.2  Biochemical indicators of vitamin D sufficiency, insufficiency and         deficiency ........................................................................................................ 16 Table 1.3  Summary of the proportions of children given supplements in different         studies.............................................................................................................. 25 Table 1.4   Summary of vitamin D intakes in children in Canada .................................... 26 Table 1.5  Summary of vitamin D intake in children in USA.......................................... 27 Table 1.6  Summary of vitamin D status of children in Canada ...................................... 28 Table 1.7  Summary of vitamin D status of children in the U.S ...................................... 29 Table 3.1  Subject characteristics ..................................................................................... 42 Table 3.2  Child anthropometric measures ...................................................................... 43 Table 3.3  Energy intakes (kcal/day) estimated using FFQ and 24 hour dietary recalls .. 44 Table 3.4  Daily dietary vitamin D intake and vitamin D intake as ?g/1000 kcal from foods estimated using the FFQ, single 24 hr recall and three 24 hr recalls. ... 47 Table 3.5  Dietary vitamin D intake (?g/day) and proportion of total intake obtained from different foods ........................................................................................ 48   ix Table 3.7  Vitamin D intake as ?g/day from foods in children given or not given vitamin D containing supplements .................................................................. 51 Table 3.8  Total vitamin D intake from diet and supplements or diet alone estimated using the FFQ and three 24 hr recalls for children given or not given             supplements. .................................................................................................... 52 Table 3.9   Proportion of total daily vitamin D obtained from supplements, fortified food and natural food sources assessed by FFQ ............................................. 53 Table 3.10  Effect of supplement use and sex on plasma 25 (OH)D in children. ............. 58 Table 3.11  Vitamin D intake from diet and supplements during different seasons ......... 59 Table 3.12  Percentage of children classified as vitamin D sufficient, insufficient and deficient, year round and during different seasons ......................................... 60 Table 3.13  Plasma 25 (OH)D as nmol/L for supplement users and non users divided into seasons ..................................................................................................... 62            x List of figures Figure 1.1       Production of vitamin D3 and D2 ....................................................................... 6 Figure 1.2       Schematic of vitamin D metabolism ................................................................. 9 Figure 3.1       Study flow chart .............................................................................................. 39 Figure 3.2       Inter-individual correlation between energy intake estimated using the FFQ and a single 24 hr recalls, and between energy intake estimated using the FFQ and three 24 hr recalls ............................................................................. 45 Figure 3.3       Amount of vitamin D consumed in natural food sources, fortified foods and supplements with comparison to the EAR and RDA ............................... 54 Figure 3.4       Proportion of children with intakes of vitamin D from diet or diet plus supplements with vitamin D intakes below the EAR or RDA for       vitamin D......................................................................................................... 55 Figure 3.5       Proportion of children given or not given vitamin D supplements with a total intake of vitamin D below or above the EAR or RDA ........................... 57 Figure 4.1       Children with vitamin D intakes below or above EAR (10 ?g/day) or RDA (15 ?g/day) with a plasma 25 (OH)D above or below 40 nmol/L or 50 nmol/L in winter months ................................................................................. 75     xi List of abbreviations  1,25(OH)D   1,25-dihydroxyvitamin D 25 (OH)D  25-hydroxyvitamin D AI    Adequate intake ANOVA  Analysis of variance BMC   Bone mineral content BMD    Bone mineral density BMI    Body mass index CCHS 2.2  Canadian community health survey cycle 2.2  CHMS   Canadian health measures survey CI   Confidence interval CNF   Canadian nutrient file CPS   Canadian paediatric society CYP P450  Cytochrome P450 CYP2R1  Cytochrome P450 2R1 CYP27B1  Cytochrome P450 27B1  D2   Ergocalciferol D3   Cholecalciferol DBP    Vitamin D binding protein DIN   Drug identification number    DRI    Dietary reference intakes EAR   Estimated average requirement FFQ    Food frequency questionnaire FGF23   Fibroblast like growth factor 23 IOM    Institute of medicine IU    International units HPLC-MS/MS High performance liquid chromatography-tandem mass spectrometry PTH    Parathyroid hormone RDA    Recommended dietary allowance SD    Standard deviation SES    Socioeconomic status SPE   Solid phase extraction SPF   Sun protection factor UL    Tolerable upper level of intake USDA   United states department of agriculture UV    Ultraviolet          xii Acknowledgements  I owe my deepest gratitude to my supervisor, Dr. Sheila Innis, for giving me this opportunity and for the invaluable assistance, direction, support and time she has spent with me over the last two years, without which I would have been unable to complete this degree. I would also like to thank my supervisory committee members, Dr. David Kitts and Dr. Tim Green for their support and guidance throughout my graduate studies.  I would like to acknowledge my fellow graduate students at the Innis lab, especially Kelly Mulder for her support and assistance throughout the program, and the tremendous amount of work she has dedicated to this project, this would not have been possible without her. I also would like to acknowledge Alejandra Wiedeman for her academic and emotional support. I would also like to thank Roger Dyer for his assistance with the laboratory analyses as well as his patience and diligence in answering even the most insignificant questions. Lastly, I would like to thank my family for their unconditional love and support.       1 Chapter 1: Literature review 1.1 Introduction Vitamin D is a fat soluble vitamin responsible for maintaining calcium and phosphorous homeostasis in the body, and is important for bone health.  Recent interest in non-skeletal functions of vitamin D has arisen following the discovery of proteins (enzymes and receptors) related to vitamin D metabolism in several tissues throughout the body not involved in bone metabolism. Thus, interest in vitamin D?s function beyond bone health has increased 1,2.    New vitamin D intake recommendations were published in 2011 by the Institute of Medicine (IOM). These recommendations included cut-off values for serum (or plasma) 25 hydroxy vitamin D (25 (OH)D), as a biochemical marker of vitamin D deficiency, insufficiency and sufficiency. The IOM (2011) evaluated that, at the time of the development of the vitamin D intake recommendations, there was not enough evidence to support the development of dietary recommendations based on health outcomes other than bone 3. Previous studies have indicated that vitamin D intakes of Canadian children are low, despite fortification of milk and margarine with vitamin D 4. Few studies on vitamin D status in children below the age of 6 years were available at the time of the dietary development of the vitamin D recommendations, thus the cut-off values for 25 (OH)D and marker of vitamin D status set by the IOM (2011) were based mainly on studies using adults and adolescents. Currently, little information is available on whether vitamin D intakes below the recommendations in young children do result in biochemical evidence of vitamin insufficiency or deficiency. Some studies have suggested that the recommended vitamin D intakes for young children may be set too high, as few children with vitamin D intakes below the EAR appear to be vitamin D insufficient 5,6.  Thus the main objectives of this thesis were to estimate vitamin D intake and the major dietary sources of vitamin D in young children in Vancouver. In addition, we sought to assess vitamin D status and determine the influence of low vitamin D intake, and the effect of season on   2 biochemical measures of vitamin D sufficiency, insufficiency and deficiency.  The following chapter will give a brief review of vitamin D sources, metabolism of vitamin D and its physiological roles. This is followed by a review of the criteria used to define vitamin D sufficiency, insufficiency and deficiency. This chapter will then provide a review of current knowledge on vitamin D intake and vitamin D status in Canadian and US children below the age of 6 years.   1.2 Vitamin D sources  Vitamin D is a fat soluble vitamin, and is considered a steroid hormone. Vitamin D can be obtained  both from the diet and by cutaneous synthesis, and as a result vitamin D status is determined by both factors 7. Individuals living in regions of the world at high latitudes rely primarily on dietary sources of vitamin D to fulfill vitamin D requirements during winter months, due to limited opportunity for endogenous synthesis in the skin during these months8,9. Thus, obtaining vitamin D from dietary sources is considered important for maintaining adequate plasma vitamin D concentrations. In the following, sources of vitamin D, including natural sources, fortified foods and fortification practices, and supplemental vitamin D will be discussed followed by a brief overview of cutaneous synthesis of vitamin D.    1.2.1 Diet, supplements, fortification and cutaneous synthesis  Vitamin D is provided in the diet mainly as cholecalciferol (Vitamin D3), which is of animal origin. Vitamin D can also be found as ergosterol (Vitamin D2) which occurs in small amounts in plant foods1. Both forms of vitamin D are produced industrially and may be used in supplements or used for fortification of foods 8,10 . Naturally occurring sources of vitamin D, however, are few, and these include foods such as fatty fish, organ meats and egg yolk.      3  Food containing naturally occurring vitamin D appear to be eaten infrequently in many westernized countries including Canada 4,11,12. Hill et al (2012) reported that only 2 % (0.09 ?g/day) of the daily vitamin D intake of 4025 Canadians was provided by eggs, and 10 % was provided by fish corresponding to 0.43 ?g /day 12. Fatty fish and egg yolk are foods not frequently eaten by many individuals in westernized North American diets, perhaps stemming in part from health concerns over the high cholesterol content of egg yolks 13 and mercury contamination in fish 14,15. The relatively low intake of foods naturally rich in vitamin D in modern westernized diets suggests that obtaining adequate amounts of vitamin D without food fortification or supplements is difficult. Fortification of foods with vitamin D in Canada in the 1940?s and 1950?s was initiated due to a high incidence of rickets among children 16. However, fortification practices were not well regulated and parts of the population had intakes too high while others had intakes considered too low, thus the regulation of the early fortification was changed. This meant that evaporated milk and milk powder was allowed added vitamin D, but fluid milk was not. Subsequently, the occurrence of rickets began to increase and the Canadian government once again allowed fortification of fluid milk in 1965 16.  Currently, milk and margarine in Canada must contain approximately 2.5 ?g vitamin D per cup (250 mL) and 13.0 ?g vitamin D/100 g, respectively 17 (Table 1.1). In addition, goats? milk, plant based milk alternatives and calcium-fortified orange juices may be fortified with vitamin D. Cheese and yogurt are permitted to be produced from vitamin D fortified milk, but vitamin D is prohibited from being added directly to the product 18.         4 Table 1.1 Vitamin D content in foods Food  Serving size Vitamin D ?g/ serving  Vitamin D ?g/100g Fortified Milk 250 mL 2.5 1.0 Fortified orange juice  250 mL 2.5 1.0 Fortified plant beverages  250 mL 2.5 1.0 Fortified margarine    10 g  1.3 13.0 Egg yolk  1  0.8 4.7 Whole egg 1  0.7 1.3 Beef liver, fried 75 g 0.9 1.2 Tuna, bluefin, cooked  75 g 5.5 7.3 Salmon Sockeye, canned/bone   75 g 13.9 18.6 Salmon, Atlantic, baked/broiled  75 g 5.1 6.8 Canned tuna, in water 75 g 0.9 1.2 Cod, Atlantic, baked/broiled 75 g 0.9 1.2 Data is derived from the Canadian nutrient file (CNF) 19.  Data collected by the Canadian community health survey (CCHS) showed that fortified milk was the largest contributor to vitamin D intake in the Canadian population, providing 49.1 % of the daily intake of vitamin D. However, despite fortification of milk, the total dietary vitamin D intake of Canadians appears to be low, providing an average total vitamin D intake from foods of 5.8 ? 0.9 ?g/day 4. Vitamin D can also be obtained from supplements, however, their consumption by only a segment of the population means that concerns for low vitamin D intakes remains 20.     5 In addition to obtaining vitamin D from the diet, vitamin D can be synthesized endogenously from 7?dehydrocholesterol which is a derivate of cholesterol, found in the skin 21. When exposed to ultra violet (UVB) light (~290 to 320 nm), the ?-steroid ring of 7-dehydrocholesterol is broken and thereby converted into previtamin D3. Subsequently previtamin D3 undergoes isomerization converting previtamin D3 into Vitamin D3 (Figure 1.1) 1,7,10,22.  The industrial production of vitamin D2 resembles that of the cutaneous synthesis of vitamin D3. First the vitamin is exposed to irradiation whereupon isomerization occurs resulting in production of vitamin D2. Vitamin D2 and D3 differ only in their side chain (Figure 1.1); their metabolism which is the same, will be reviewed in the following section 1,23.                         6 Figure 1.1 Production of vitamin D3 and D2  A     B    A) Conversion of 7-dehydrocholesterol to vitamin D3. B) Conversion of ergosterol by irradiation to vitamin D2. The B-ring of 7-dehydrocholesterol and ergosterol is broken and both form the pre-form of the vitamin and both subsequently undergo isomerization to form vitamin D3 and D2. Figure modified from Bikle (2009) 23.        7-dehydrocholesterol  Previtamin D3  Vitamin D3 UVB Isomerization  Ergosterol   Previtamin D2 Vitamin D2 Isomerization Irradiation   7 1.3  Vitamin D metabolism Vitamin D obtained in the diet or synthesized in the skin are both transported to the liver, where they are metabolized identically 1,7.  The following section provides a brief review of absorption and transport of vitamin D obtained from the diet as well as vitamin D derived from endogenous synthesis. This is followed by a description of the hepatic and renal metabolism of vitamin D from the diet and endogenous synthesis. Finally, the physiological roles of vitamin D in bone health and emerging evidence of non-skeletal functions of vitamin D are discussed as background to the research in this thesis.    1.3.1 Absorption and transport Vitamin D is a fat-soluble vitamin, thus vitamin D in the diet is best absorbed in the presence of fat.  After ingestion, dietary vitamin D is incorporated into micelles in the intestine and subsequently it diffuses into the intestinal enterocytes. Within the enterocytes, the vitamin is incorporated into chylomicrons, which enter the lymphatic system and thence enters the blood 24. Vitamin D is transported in the blood either by chylomicrons to the liver and extra hepatic tissues or bound to the vitamin D binding protein 1,23. After synthesis in the skin, cholecalciferol diffuses into the blood where it also binds to vitamin D binding protein (DBP) for transport to the liver or extra hepatic tissues. Excess vitamin D3 can be stored in adipose tissue for months to years and released when little vitamin D is synthesized in the skin 1,7,21. In the liver, the metabolism of D3 and D2 obtained from the diet, released from storage in adipose tissue, or vitamin D synthesized in the skin follow the same path of hydroxylation 1.   1.3.2 Hydroxylation of vitamin D in the liver and kidney Vitamin D must undergo two hydroxylations before it becomes biologically active. In the liver, cholecalciferol is converted into 25- hydroxyvitamin D (25 (OH)D) by 25- hydroxylase,   8 which is thought to be cytochrome P450 2R1 (CYP2R1). Most of the newly synthesized 25 (OH)D is secreted into the blood where it binds to vitamin D binding protein (DBP), and has a half-life of approximately 15 days to three weeks 1,25. For the vitamin to become active, 25 (OH)D must undergo a second hydroxylation, which is initiated if there is a decrease in the calcium concentration in the blood. This decrease is detected by calcium sensing proteins in the thyroid gland, which in response secretes parathyroid hormone (PTH). The increase in PTH leads to up-regulation of 1?-hydroxylase (CYP27B1) in the kidney which in turn leads to synthesis of 1,25 (OH)D also called calcitriol, by hydroxylation of 25 (OH)D. Calcitriol is thus under tight homeostatic control and has a short half-life of a few hours 1,7,10,25. Inactivation and excretion of vitamin D is believed to be initiated by 24-? hydroxylase (CYP24), which is up-regulated by increased levels of calcitriol itself and fibroblast like growth factor 23 (FGF23) 23. Through a series of steps, CYP24 converts the vitamin D metabolites 25 (OH)D and 1,25 (OH)D by 24-hydroxylation 7,23. This leads to the formation of calcitroic acid as the end product, which is secreted with the aid of bile acids, ultimately being excreted in the feces 1,7,23.    1.3.3 Physiological roles of vitamin D Vitamin D is known to function through its actions on the intracellular vitamin D receptors (VDR), which influences gene transcription and thus protein synthesis. The best known actions of the proteins synthesized in response to binding of vitamin D to the VDR are those involved in the regulation of serum calcium and phosphate homeostasis 1,7. The homeostatic control of calcium and phosphate is regulated by two counteracting hormones, parathyroid hormone (PTH) and calcitonin, which are further described in the following section. Additionally, numerous studies have reported evidence that vitamin D has several positive effects on non-skeletal health outcomes. An overview of these new emerging areas of vitamin D are briefly reviewed in this section.    9 1.3.3.1 Calcium and phosphate homeostasis and bone health The best known roles of calcium and phosphorous in the body are in bone mineralization. However, calcium has numerous other essential functions, including blood clotting, nerve conduction, muscle contraction, enzyme regulation, and membrane permeability 1. Many of these roles are reliant on a stabile calcium concentration in the blood, thus the regulation of calcium is vital. The main role of vitamin D is to regulate and maintain the calcium blood concentrations 7. Schematic of vitamin D metabolism is shown in Figure 1.2.  Figure 1.2 Schematic of vitamin D metabolism   Blood Ca2+?1,25 OHD? Ca 2+ absorption25-OHD  in bloodVitamin D from dietVitamin D from        sun exposure? PTH?1,25 OHD? Blood Ca2+? Bone calcification? Bone calcium resorption? PTH?1,25 OHD?PTH?Calcitonin Schematic illustration vitamin D?s metabolism and regulation of calcium homeostasis. Modified from Holick (2007)10.   10 As previously introduced, when blood concentrations of calcium decrease, the parathyroid gland detects the decline and secretes PTH. PTH in turn activates 1?-hydroxylase and 1,25 (OH)D is synthesized. Calcitriol and PTH exerts their functions on three main target tissues which are the intestine, kidneys and bone, ultimately leading to increased calcium or phosphate concentrations in the blood. Vitamin D can act either by influencing gene expression or as a steroid hormone 7.  In the intestine, calcitriol functions to increase calcium or phosphate absorption, by affecting gene expression and thereby initiating transcription of specific proteins involved in calcium absorption. Briefly, gene expression is initiated by 1,25 (OH)D binding to a nuclear vitamin D receptor (VDR). This complex is thought to bind to retinoic acid X receptor to form a heterodimeric complex, which in turn can interact with vitamin D response elements (VDREs) found on specific DNA sequences, ultimately resulting in regulation of gene expression, and thereby either enhancing or inhibiting transcription 1,7,21. As an example, calbindin D9k is a calcium binding protein found in the intestine which is synthesized in response to 1,25 (OH)D. After synthesis, calbindin functions on the brushborder to facilitate calcium absorption from the intestine.  Additionally, it is believed that 1,25 (OH)D induces expression of epithelial cell calcium channels called epithelial calcium transient receptor potential vanilloid-type family member 6 (TRPV6) which are found on the brush border membrane of the intestine. The TRPV6 receptor interacts with calbindin D9k to increase calcium absorption in the intestine. Further, when calcium concentrations in the blood decrease, 1,25 (OH)D and PTH appear to stimulate calcium reabsorption in the kidney, thus decreasing the excretion of calcium 26.  Calcitriol and PTH also play a role in mobilization of calcium and phosphate from the bone. Calcitriol or PTH also facilitates the formation and activation of osteoclasts by interacting with osteoblasts which in turn induces expression of receptor activator of nuclear factor-kB ligand (RANKL). RANKL then interacts with immature osteoclasts which induce maturation of   11 the osteoclasts that are responsible for mobilization of calcium and phosphate from the bone, causing calcium levels to rise. During times of vitamin D deficiency, calcium absorption from the intestine is decreased and mobilization of calcium from the bone is increased 1,7,21. Conversely, high serum levels of calcium or phosphorous lead to down-regulation by the hormone calcitonin which is secreted by the thyroid gland. Calcitonin promotes bone mineralization and blocks calcium mobilization from the bone 1.  Further, calcitriol is thought to suppress PTH production and secretion when interacting with its receptor (VDR). Fibroblast like growth factor 23 (FGF23) is another protein involved in calcium and phosphorous homeostasis. It is produced in the osteocytes and osteoblasts in response to elevated levels of calcitriol and phosphate. It acts on the parathyroid gland to decrease PTH production. Further, FGF23 reduces CYP27B1 expression and increases expression of renal CYP24, thus decreasing 1,25 (OH)D synthesis and increasing the production of inactive metabolites of vitamin D. Further, FGF23 decreases the reabsorption and increases excretion of phosphate in the kidney by decreasing the expression of the sodium-phosphate co-transporter 1,7,23.     1.3.3.2 Emerging roles of vitamin D Recent interest in non-skeletal functions of vitamin D has arisen following the discovery of vitamin D receptors (VDR), vitamin D responsive elements (VDREs), vitamin D metabolites and enzymes related to vitamin D metabolism in several tissues throughout the body not involved in bone metabolism. These tissues include the immune system, brain and placenta 27?30.  Briefly emerging evidence relating vitamin D to immune function has found VDRs are present in several different cells of the adaptive and innate immune system, of which many are able to locally synthesize 1,25 (OH)D. It is believed that 1,25 (OH)D enhances the activity of the innate immune system and  decrease the adaptive immune system 31, with 1,25 (OH)D also shown to enhance phagocytosis and modulate activated T and B lymphocytes.  Experimental   12 studies have also indicated that 1,25 (OH)D is present in the cerebrospinal fluid of humans and can cross the blood brain barrier. Further, CYP27B1 and CYP24A1 have found to be present in the fetal and adult brain, thus 1,25 (OH)D can be produced locally in these cells or be  inactivated 28. However, little is as yet known about vitamin D and brain development or function in human. Similarly, other studies have found CYP27B1 is present in the human placenta which is also capable of synthesizing 1,25 (OH)D and 24,25 (OH)D. It is believed that 1,25 (OH) D promotes anti-bacterial and anti-inflammatory responses and thus may function in placental immune and inflammatory response 31.  1.4 Consequences of low vitamin D status During vitamin D deprivation, calcium absorption from the intestine and reabsorption in the kidney is decreased. This in turn prompts blood PTH concentrations to remain elevated and calcium mobilization from the bone persists. Thus, chronic low vitamin D status can cause bone demineralization and poor mineralization. Ultimately, vitamin D deficiency can lead to rickets in children, which is characterized by continuous growth of cartilage but failure to mineralize the bone 8. Adequate vitamin D status is especially important for children to ensure proper mineralization of bones 21.    1.5 Factors affecting vitamin D status In addition to vitamin D intake, several factors can influence an individual?s vitamin D status. These factors include physical individual factors and environmental factors, including lifestyle choices, which will be discussed next.   1.5.1 Individual factors affecting vitamin D status A number of individual factors can affect vitamin D status. Studies have found that obese individuals have lower 25 (OH)D concentrations than individuals considered to be normal   13 weight. This is believed to be caused by sequestering of vitamin D in the adipose tissue, but little is known about the cause and effect 32.  Additionally, it is recognized that skin pigmentation affects the cutaneous synthesis of vitamin D. Darker skin types have more melanin which is believed to act as a natural sunscreen, and thereby decrease the amount of vitamin D synthesized in the skin 9. Ageing also has an effect on cutaneous synthesis of vitamin D, as the amount of 7-dehydrocholesterol in the skin decreases with age. Therefore, elderly individuals may need longer sun exposure than younger individuals to achieve the same effect 9.    1.5.2 Environmental and lifestyle factors affecting vitamin D status The cutaneous synthesis of vitamin D can also be affected by environmental factors as well as lifestyle factors. At higher latitudes, the intensity of the UVB rays reaching the earth?s surface is reduced during winter months, due to the earth?s inclination. Consequently, cutaneous synthesis of vitamin D is greatly reduced or does not occur 9,22. Further, overcast days during the year and air pollution are also believed to decrease the amounts of vitamin D synthesized in the skin 33. Thus, the availability of adequate vitamin D from the diet becomes particularly important, especially in areas of the world located at higher latitudes such as Vancouver, which is located at latitude of 49?N 16?. Vancouver also has few hours of sunshine during winter and fall months namely, 60, 85, 134,182, 231 and 229 hours of sunshine for the months of January - June and 294, 268, 199, 125 64 and 56 h/month for the 6 months of July - December, respectively 34. Additionally, concerns over skin cancer have lead to an increased use of sunscreen, sun avoidance by coverage of the skin with clothing or staying indoors 9,35,36. Use of sunscreen with sun protection factor (SPF) 15 and higher is believed to block nearly all cutaneous synthesis of vitamin D if applied as recommended 2. Further, increasing indoor activities, work and school also reduce sun exposure. Canadian children have physical activity levels below the recommendations of 60 min/day 37,38, thus out-door activity in children may   14 also have decreased. In addition, studies suggest that time spent watching TV and playing computer games have increased in children, possibly also leading to decreased time spent       out-doors 39.   1.6 Assessment of vitamin D status The best biomarker for vitamin D status is considered to be 25 (OH)D, as it is not under homeostatic control and has a relatively long half-life of approximately 15 days. Circulating levels of 25 (OH)D reflects the sum of vitamin D synthesized cutaneously and that obtained from the diet and supplements. Conversely, calcitriol is under homeostatic regulation and has a short half-life which limits its usefulness as a biomarker for vitamin D status. PTH has also been considered as a biomarker of vitamin D status due to the inverse relationship between PTH and 25 (OH)D. During a state of vitamin D deficiency, PTH remains elevated as the intestinal absorption of calcium and renal calcium reabsorption is decreased, which means that mobilization of calcium from the bone is needed to maintain circulating calcium concentrations. According to Prentice et al (2008), there is a wide variation of PTH concentrations within and among individuals, and several factors such as demographics, physiological factors, and other dietary variables can affect PTH levels. As a result, defining a normal PTH concentration has been difficult.    1.6.1 Vitamin D sufficiency, insufficiency and deficiency The most recent dietary recommendations for vitamin D and calcium from the Institute of Medicine (IOM) were published in 2011 3. The 2011 Dietary Reference Intakes (DRI) for vitamin D noted that 25 (OH)D cut-points specifying vitamin D status had not undergone a systematic evidence based development process, and that several different cut-off levels were used by different expert groups. The recommendations for vitamin D that the IOM (2011)   15 developed are based on bone health as the sole outcome and do not include any roles of vitamin D other than skeletal health outcome, as they evaluated that the evidence base for other health outcomes were inconclusive. The IOM (2011) considered that there is evidence to believe that an increased risk of rickets, impaired calcium absorption, and decreased bone mineral density (BMD) occurs at serum 25 (OH)D concentrations below approximately 30 nmol/L. They concluded that maximal calcium absorption occurs at 25 (OH)D of 30 to 50 nmol/L, and that there is no evidence to suggest further benefits for bone health when 25 (OH)D increases above 50 nmol/L. Based on this, it is assumed that vitamin D intakes which achieve circulating levels of 40 nmol/L 25(OH)D is equivalent to the median requirement of the population, and that intakes that achieve circulating levels of 50 nmol/L would cover the needs of 97.5 % of the population. Due to the difficulty in estimating the amounts of vitamin D an individual will obtain from sun exposure, the vitamin D intake recommendations proposed by the IOM (2011) were developed assuming minimal sun exposure 3.  Several experts disagree with the 25 (OH)D cut-offs for vitamin D sufficiency, insufficiency and deficiency suggested by the IOM (2011).  Holick (2007) argued that 25 (OH)D concentrations of 75 nmol/L should be considered sufficient because PTH and 25 (OH)D are inversely correlated until PTH begins to plateau at a 25 (OH)D of  approximately 75 - 100 nmol/L, and intestinal calcium absorption is significantly increased at between 50 and  80 nmol/L 40. Similarly, Vieth (2011)  advocated defining vitamin D sufficiency at 25 (OH)D >75 nmol/L 41. The Canadian Paediatric Society (CPS) defines vitamin D sufficiency, insufficiency and deficiency based on similar considerations to Holick (2007) and Vieth (2011). The CPS suggested that bone mobilization, PTH production and intestinal calcium absorption is stabile at plasma 25 (OH)D between 75 nmol/L to 225 nmol/L 42.    16 As shown in Table 1.2, utilizing the Canadian Paediatric Society?s (2007) classifications will identify a higher prevalence of individuals with vitamin D insufficiency than when using the cut-offs recommended by the IOM (2011). The Endocrine Society has suggested different cut-off for 25 (OH)D in children aged 4 ? 8 years. The Endocrine Society has suggested that plasma 25 (OH)D below 50 nmol/L should be classified as deficiency and plasma 25 (OH)D concentrations above 75 nmol/L should be considered sufficient, and the Endocrine Society also suggested a daily vitamin D intake requirement for children age 4 ? 8 years of 15 ? 25 ?g/d.  In this thesis, vitamin D status will be defined based on the cut-off levels established by the IOM (2011), although the discussion will include commentary on the proportion of children meeting the criteria for insufficiency using the Canadian Paediatric Society (2007) and Endocrine Society cut-off values.  Table 1.2 Biochemical indicators of vitamin D sufficiency, insufficiency and deficiency Definition IOM CPS ES Deficient < 30 < 25 < 50 Insufficient 30 ? 50 25 ? 75 52.5 ? 72.5  Sufficient/Optimal 50 ? 125 75 ? 225 75- 250   Potential adverse effects > 125 > 225 -  Values are nmol/L 25 (OH)D. Institute of medicine (2011), Canadian Paediatric Society (2007), Endocrine Society (2011).   1.7 Vitamin D intake recommendations As introduced in section 1.5, the amounts of vitamin D synthesized in the skin can vary considerably among individuals and within individuals at different times of the year. The IOM committee reviewed studies that investigated the dose-response relationship between total   17 vitamin D intake and plasma 25 (OH)D under conditions of no or minimal sun exposure. They concluded that an intake of 10 ?g/day (400 IU) and 15?g/day (600 IU) on average achieved a 25 (OH)D of 59 nmol/L and 63 nmol/L, respectively.  Due to uncertainties such as inter-study variances and comparability of results from studies using different 25 (OH)D assay methods, the committee estimated that intakes10 ?g/day (400 IU) and 15 ?g/day (600 IU) would achieve a 25 (OH)D of 40 nmol/L and 50 nmol/L respectively 3. The vitamin D intake recommendations for children aged 4 - 8 years were extrapolated from studies in older children and adolescents and are based on measures of bone health 3.  1.8 Current knowledge of vitamin D intake and status in children At the time that the research in this thesis was designed and started, there was a small amount of information on vitamin D intake in children, but information on vitamin D status of groups of Canadian 5 - 6 year olds was limited. The following section provides a summary of current information on vitamin D in children from two national surveys in Canada and the NHANES survey in the U.S. Further, research studies addressing vitamin D intake or status in young children in Canada and the U.S will be reviewed.  1.8.1 Canadian community health measures survey cycle 2.2 - 2004 The 2004 Canadian community health measures survey cycle 2.2 (CCHS 2.2) was a cross-sectional study that collected nutritional information from a nationally representative sample of 34,789 Canadians of whom 5655 were children aged 1 - 8 years. The dietary information collected was based on a single computer assisted 24-hour recall from 31,107 participants. A second recall was collected from 10,786 participants, which constituted about 30 % of the sample population 4. Based on the initial recall, Canadian children had a mean intake of   18 vitamin D in 2004 of 6.2 ? 0.1 ?g/day and a median intake of 5.6 (IQR 4.1 ? 7.5) ?g/day 4. The 75th percentile of intake was 7.5 ?g/day, indicating that at least 75 % of the children consumed an amount of vitamin D below the EAR of 10.0 ?g/day, as defined by the IOM (2011). The main source of dietary vitamin D for this age group was fortified milk, which contributed 75 % of the vitamin D intake 4. When the intake from supplements and diet was combined, the mean and median intake of vitamin D was 9.5 ? 0.2 ?g/day and 8.2 (IQR 5.4 ? 13.2) ?g/day in children aged 4 ? 8 year old children, respectively 43. The CCHS 2.2 did not collect blood samples from the population and thus vitamin D status could not be assessed.   1.8.2 Canadian health measures survey 2007 - 2009 and 2009 - 2011 The Canadian health measures survey (CHMS 2007 ? 2009) is a nationally representative survey of 5306 Canadians in which blood samples were collected and vitamin D status was measured. However, no children under the age of 6 were included in CHMS (CHMS 2007 - 2009). The 2007 ? 2009 national survey showed a mean 25 (OH)D of 75.0 nmol/L and indicated that at the time, 95.6 % of 6 - 11 year olds had plasma 25 (OH)D concentrations above 37.5 nmol/L and 48.6 % had 25 (OH)D above 75 nmol/L 44. Fourteen percent (14.1 %) of the children within this age group had 25 (OH)D concentrations below 50nmol/L 20. Comprehensive dietary collection was not part of the CHMS, but information such as frequency of consumption of different food groups was collected 20. The survey showed that 28.7 % of the children were taking a vitamin D containing supplement, however, no information on frequency of the consumption or dose was available 20.  Recently, some data from the CHMS Cycle 2 (2009 ? 2011) was published 45. This cycle included children aged 3 ? 5 years which the previous cycle had not. Of the 3 ? 5 year old children, 518 children provided a blood sample46 and the mean plasma 25 (OH)D reported for the 3 - 5 year olds was 73.9 nmol/L and it was estimated that 11.0 % of the 3 ? 5 year olds had a 25 (OH)D below 50 nmol/L. Of the 6 ? 11 year olds, in the CHMS   19 (2009 ? 2011), 974 provided a blood sample and 24.0 % of the children were found to have a plasma 25 (OH)D below 50 nmol/L. The mean 25 (OH)D was 67.3 nmol/L, and boys had a higher 25 (OH)D than girls of 72.0 nmol/L versus 63.0 nmol/L, respectively 45,47.     1.8.3 USA ? National health and nutrition examination survey The National Health and Nutrition Examination Survey (NHANES 2005 - 2006) is a national representative sample of the U.S population, which has been ongoing since the 1960?s and several series of this national population study have been conducted 48. The NHANES 2005 - 2006 estimated dietary and supplemental intake of vitamin D for the U.S population using two 24 hour recalls and a questionnaire, respectively. The available data published on vitamin D intake and status from the NHANES is summarized here. The 2005 - 2006 NHANES reported a mean vitamin D intake from diet alone for boys and girls 4 ? 8 year olds of 6.4 ? 0.3 and 5.5 ? 0.3 ?g/day, respectively. When including intake from supplements, the mean total intake of vitamin D for boys and girls aged 4 - 8 years increased to 9.3 ? 0.4 and 7.9 ? 0.6 ?g/day, respectively. Supplements were given to 43 % of the boys and 34 % of the girls aged 4 ? 8 years. The mean daily intake from supplements in children taking supplements was 6.6 ? 0.4 ?g/day for boys and 7.9 ? 1.3 ?g/day for girls49.   Vitamin D status was reported for the NHANES 2001 - 2006 and this included 904 boys and 895 girls aged 1 - 5 years. The mean 25 (OH)D concentration was 71.0 nmol/L in boys and  70.0 nmol/L in girls. Of the total sample, 14 % was reported to have 25 (OH)D below 50 nmol/L and 63 % below 75 nmol/L 50. This estimate of 14 % of U.S children 1 ? 5 years of age in 2001 ? 2006 with a 25 (OH)D < 50nmol/L is similar to the estimate of 11 % of Canadian children 3 ? 5 years of age in the 2009 ? 2011 CHMS with a 25 (OH)D < 50 nmol/L 47.       20 1.8.4 Research reports At the time the research in this thesis was designed in 2010, little information had been published on the vitamin D status of groups of Canadian children under the age of 6 years. The following section provides a review of recent studies on vitamin D intake and status of children in Canada and the U.S, other than those using national survey data.   1.8.4.1 Canada In 2005, Roth et al measured 25 (OH)D in 68 children seen in April 2003 in Edmonton, which is at latitude 52?N. Of the 68 children aged 2 - 16 years, 35 of them were aged between 2 and 8 years. The mean 25 (OH)D for the 2 ? 8 year olds was 51.5 nmol/L with an SD of 14.6 nmol/L. Of the 2 - 8 year olds, 2.9 % had a 25(OH)D below 25 nmol/L, but this represented only one child. However, 17 % (n= 6) had 25 (OH)D concentrations below 40 nmol/L, although the sample size of 35 children was small. For the entire group of 68 children, 34 % (n= 23) had a 25 (OH)D below 40 nmol/L and 5.9 % (n= 4) had a 25 (OH)D below 25 nmol/L. Of the entire group, 27 % were reportedly using a multivitamin regularly. The median vitamin D intake in the 2 ? 8 years olds was 8.3 (IQR 4.6 ? 10.4) ?g/day with a lower intake of 5.8 (IQR 2.4 ? 8.2) ?g/day estimated using a FFQ and single 24 hr recall, respectively51.  Hayek et al (2010) conducted a study including 388 Inuit preschoolers age 3 ? 5 years living at latitudes between 51?N ? 70 ?N. They showed a daily mean vitamin D intake of 6.6 ? 2.9 ?g/day (n= 279). Supplement use was recorded, as well as the frequency of supplement use and this showed that 3.7 % of the children were given a vitamin D supplement (presumably alone) and 16.8 % were given a multivitamin. Blood was collected from 282 children in summer and from 52 different children in winter (February ? April). The median 25 (OH)D during summer was 48.3 (IQR 32.8 ? 71.3) nmol/L. The children (n= 52) who were included in the study during winter had a median 25 (OH)D of 37.8 (IQR 21.5 ? 52.0) nmol/L. This   21 corresponded to 51.7 % and 72.8 % of the children having 25 (OH)D below 50 nmol/L in summer and winter, respectively. In addition, vitamin D insufficiency was assessed using the Canadian Paediatric Society?s cut-off of (< 75.0 nmol/L), which classified 78.6 % and 96.8 % of children as insufficient during summer and winter, respectively 52.   A more recent study by Hayek et al (2013) conducted between June 2010 and June 2011, assessed vitamin D status and intake in 508 children 2 - 5 year of age in Montreal, which is at latitude 45?N. They found that 88 % of the children had a 25 (OH)D ? 50 nmol/L and 49.4 % of the children had plasma 25 (OH)D  ? 75nmol/L. However, 95 % of the children had vitamin D intakes below the EAR of 10 ?g/day. The median dietary vitamin D intake was 5.9 (IQR 3.8 ? 8.0) ?g/day based on a single 24 hr recall. Approximately 28 % of the children were reportedly given supplements, estimated using a FFQ, with a median dose of 7.1 (IQR 3.2 ? 10.0) ?g/d in children given supplements. The total vitamin D intake from diet and supplements in all children was 9.9 (IQR ? 7.1 -13.2) ?g/day based on the FFQ 6.  Another recent Canadian study had similar findings. Maguire et al. (2013) examined vitamin D status in 1311 children aged 2 ? 5 years from Toronto, which is at latitude 43.4?N. The mean 25 (OH)D was 88 nmol/L; 35 % had a 25 (OH)D  below 75 nmol/L and 6 % had concentrations below 50 nmol/L. The study did not collect comprehensive dietary data on vitamin D intake, but did collect data on frequency of consumption of cups of milk, and frequency of use of supplements. Of the 1311 children, 61 % reported taking supplements. The mean intake of cows? milk was 455 mL/day, which would provide 4.7 ?g/day vitamin D from milk. The authors reported a 6.5 % increase in 25 (OH)D for each one cup of milk consumed.  Further, an increase of 13.2 % 25 (OH)D was attributed to daily use of vitamin D supplements. Winter was reported to result in decrease in 25 (OH)D of 10.7 % nmol/L when compared to summer. Skin pigmentation was also a major factor with a 9.9 % nmol/L difference between skin type I-III  and IV -VI on the Fitzpatrick scale 53.     22  Hill et al (2012) conducted a study on vitamin D intake and the most common food sources of vitamin D among 4025 Canadians, of which 534 were children aged 2 ? 12 years. They showed a mean intake of 4.4 ? 0.1 ?g/day from food in the 2 - 12 year olds. No data on supplement use was included and data on proportion of vitamin D provided by different food sources was only reported for the whole study population, and not by age group 12.    1.8.4.2 U.S.A Abrams et al (2012) conducted a double blind randomized controlled trial investigating whether supplementation with 1000 IU/day (25 ?g/day) would affect calcium absorption in 4 - 8 year old girls (n= 64).  The study was conducted in Texas which is located at latitude 29?N. The baseline vitamin D intake was 5.5 ? 0.2 ?g/day which did not include supplemental vitamin D, as supplement use was an exclusion criteria. The mean ? SD baseline 25 (OH)D was 69.1 ? 18.5 nmol/L and no differences in baseline characteristics were found between the randomized groups. After 8 weeks of supplementation, the 25 (OH)D in the supplement group (n= 32) had increased to 89.9 ? 25.7 nmol/L. PTH had decreased from 21.4 ? 10.4 to 12.9 ? 7.1 pg/mL, with no significant changes from baseline observed in the placebo group (n= 31). Stable isotopes were used to assess calcium absorption, however, despite the increased 25 (OH)D and decreased PTH in the supplement group, no significant effects on calcium absorption was observed.     Carpenter et al (2012) recruited 776 children aged six months to three years from Connecticut which is at latitude 41?N. They showed a mean ? SD vitamin D intake from food and supplements of 6.2 ? 3.2 ?g/day. Children given vitamin D containing supplements above 10 ?g/day were excluded from the study, which may mean the results do not reflect the general population. The children were mainly Hispanic (64 %) and African American (23 %), and had a mean ? SD 25 (OH)D of 66.0 ? 22 nmol/L, of which 15 % and 5.8 % had a 25 (OH)D below 50   23 nmol/L and 40 nmol/L, respectively. Despite classifying 15 % as vitamin D insufficient, only 2.5 % of the children showed elevations of PTH and alkaline phosphatase (ALP).   Hill et al (2012) reported vitamin D intakes for 1350 U.S children aged 2 ? 12 years. For these U.S children, the mean ? SD intake was 4.4 ? 0.1 ?g/day, which was similar to the intake data for Canadian children described in the same report. The sample was selected based on the Canadian and U.S. census statistics aiming to reflect the respective populations12.   Kemp et al (2007) collected blood samples from 142, 1 - 8 year old African American and Hispanic children from Newark (latitude 40?N). This study based on the relationship between blood lead concentrations and 25(OH)D during winter and summer, and its relation to child age and race. Blood was collected from all children in both winter and in summer, with the aim of estimating the increase in 25 (OH)D from winter to summer. The children were divided into two age groups, 1 - 3 years and 4 - 8 years. The mean 25 (OH)D for children aged 1 ? 3 was 83.2 ? 3.0 nmol/L during winter and 84.2 ? 2.7 nmol/L during summer. Children aged 4 - 8 years showed a larger seasonal difference in 25 (OH)D with concentrations of 63.0 ? 3.0 and 84.2 ? 2.7 nmol/L in winter and summer, respectively. Of the entire group of children, 12 % had 25 (OH)D below 40 nmol/L in winter and 0.7 % had 25 (OH)D below 40 nmol/L in summer 54.  In 2008, Gordon et al recruited 380 infants and toddlers aged 8 - 24 months from Boston (latitude 42?N). The population was primarily African American or Hispanic. The mean 25 (OH)D for infants and toddlers was 87.9 ? 37.9 and 85.6 ? 30.7 nmol/L, respectively. Forty percent (40 %) had a plasma 25 (OH)D below 75 nmol/L, 12.1 % were classified as deficient (defined as ? 50 nmol/L) and 1.9 % had severe deficiency (defined as a 25 (OH)D below 20 nmol/L). Forty participants were classified as deficient, and returned for a radiographic assessment of the wrist and knee. It was estimated that 32.5 % of these children showed signs of demineralization and one child showed signs of rickets 55.    24 Stein et al (2006) conducted a cross-sectional study that included 168 girls aged 4 ? 8 years to investigate the relationship between 25 (OH)D and bone area, bone mineral density (BMD) and bone mineral content (BMC). The study was conducted in Athens U.S (latitude 34?N) and included white (n= 120)  and black (n= 48) girls who were found to have a mean ? SD plasma 25 (OH)D of 99.2 ? 28.2 nmol/L and 80.4 ? 23.1 nmol/L, respectively. Only four children had a plasma 25 (OH)D below 50 nmol/L corresponding to 2.4 % of the total sample. The authors concluded that 25 (OH)D concentrations were not positively associated with BMD or BMC 56.   1.8.5 Summary The available data published over about the last decade suggests that vitamin D intake in many Canadian as well as U.S children below 6 years of age is below the current EAR of 10 ?g/day and RDA of 15 ?g/day (Table 1.4, Table 1.5). However, the limited data on vitamin D status based on measures of 25(OH)D indicates that the proportion of children with vitamin D insufficiency is much lower than the proportion of children not meeting the EAR (Table 1.6, Table 1.7). However, current data also suggest that many children are given vitamin D containing supplements, although the frequency of dose given varies considerably among different studies (Table 1.3).           25  Table 1.3 Summary of the proportions of children given supplements in different studies  Age Sex Supplement use Canada    CHMS (2007 - 2009)1 1 ? 8 Both 28.7 Hayek (2010)2 3 ? 5 Both 20.5 Hayek (2013) 2 ? 5 Both 27.7 Maguire (2013) 2 ? 5 Both 61.0 Roth (2005)3 2 ? 8 Both 27.0 USA    NHANES (2005 - 2006) 4 ? 8 Boys 43.0 NHANES (2005 ? 2006) 4 ? 8 Girls 34.0 Stein (2006)4 4 ? 8 Girls 61.0 1Data derived from Whiting et al (2011); 2 3.7 % Vitamin D containing supplement and 16.8 % multivitamin, does not specify whether they contained vitamin D; 3MVI use, does not specify whether they were vitamin D containing; 4Data reported for Caucasian girls, supplement use of black girls 32 %.     26  Table 1.4  Summary of vitamin D intakes in children in Canada N/R ? Not reported; 1Supplement data collected by questionnaire; 2Does not specify whether or not supplements have been included in the estimate.          Reference Sex Age n Vitamin D intakes ?g/d Source Collection method     Mean ? SD Median (IQR)   CCHS 2.2 (2004) Both 1 ? 8 5655 6.2 ? 0.1 5.6 (4.1 - 7.5) Foods Single 24 hr recall CCHS 2.2 (2004) Both 1 ? 8 5655 9.5 ? 0.2 8.2 (5.4 - 13.2) Food + supp Two 24 hr recalls1  Hayek et al (2010) Both 3 ? 5 275 6.3 ? 2.9 N/R Unclear2 Single 24 hr recall Hayek et al (2013) Both 2 ? 5 479 N/R 5.9 (3.8 - 8.0) Food Single 24 hr recall Hayek et al (2013) Both 2 ? 5 479 N/R 9.9 (7.1 - 13.2) Food + supp FFQ1 Hill et al (2012) Both  2 ? 12 534 4.4 ? 0.1 N/R Food 7 ? 14 diet records Roth et al (2005) Both 2 ? 8 35 N/R 8.3 (4.6 ? 10.4) Food + supp FFQ1 Roth et al (2005) Both 2 ? 8 25 N/R 5.8 (2.4 ? 8.2) Food Single 24 hr recall   27 Table 1.5 Summary of vitamin D intake in children in USA Reference Sex Age (Yrs) n Vitamin D intake ?g/d Source Collection method     Mean ? SD   NHANES  (2005 ? 2006) Boys 4 ? 8 431 6.4 ? 0.3 Foods Two 24 hr recalls NHANES  (2005 ? 2006) Boys 4 ? 8 431 9.3 ? 0.4 Food + supp Two 24 hr recalls1 NHANES  (2005 ? 2006) Girls 4 ? 8 468 5.5 ? 0.3 Food Two 24 hr recalls NHANES  (2005 ? 2006) Girls 4 ? 8 468 7.9 ? 0.6 Food + supp Two 24 hr recalls1  Abrams et al (2012)2 Both 4 ? 8 62 5.5 ? 2.0 Food Single 24 hr recall & three day diet record Carpenter et al (2012)3 Both 6 mo - 3 yrs 776 6.2 ? 3.2 Food + supp Three day diet record Hill et al (2012) Both 2 ? 12 1350 4.4 ? 0.1 Food 7 ? 14 days diet record Stein et al (2006)4 Girls 4 ? 8 114 9.7 ? 5.7 Food + supp Three day diet record1 Median values were not reported for any of the studies; 1 Supplement data collected by questionnaire; 2 Study excluded children taking supplements; 3Study excluded children taking > 10 ?g supplemental vitamin D; 4Results shown for white girls only, vitamin D intake among black girls (n= 42) were 6.8 ? 5.5 ?g/day.     28   Table 1.6 Summary of vitamin D status of children in Canada  Reference Latitude N? Sex Age (Yrs) n Vitamin D  nmol/L < 50.0  nmol/L(%) ? 75.0  nmol/L(%) CHMS (2007 - 2009) > 42? Both 6 ? 11 903 75.0 14.1 48.6 CHMS (2009 - 2011) CHMS (2009 - 2011) CHMS (2009 - 2011)1 CHMS (2009 - 2011)1 >42? >42? >42? >42? Both Both Boys Girls 3 ? 5 6 - 11 6 -11 6 ? 11 518 974 N/R N/R 73.9  67.3 72.0  63.0  11.0 24.0 N/R N/R N/R N/R N/R N/R Hayek et al (2010)2 Hayek et al (2010)3 51? - 70? 51? - 70? Both Both 3 ? 5 3 ? 5 282 52 48.3 (32.7 ? 1.4) 37.8 (21.5 ? 2.9) 51.7 72.8 78.6 96.8 Hayek et al (2013) 45.0? Both 2 ? 5 508 74.4 (60.3 ? 93.5) 10.6 49.4 Maguire et al (2013) 43.4? Both 2 ? 5 1311 88.0 6.0 35.0 Roth et al (2005) 52.0? Both 2 ? 8 35 51.5  N/A N/A N/R = not reported. Data in bold are median and data in italic are means; 1n was reported for children who provided a blood sample but the actual sample size used to estimate the mean plasma 25 (OH)D was not reported, should be interpreted with caution; 2Summer; 3Winter.     29 Table 1.7 Summary of vitamin D status of children in the U.S  Reference Latitude  N? Sex Age (Yrs) n Vitamin D nmol/L1) < 50.0  nmol/L (%) ? 75.0 nmol/L(%) NHANES (2001 ? 2006) National Boys 1 ? 5 904 71.0  14.0 39.0 NHANES (2001 ? 2006) National Girls 1 - 5  895 70.0 14.0 35.0 Abrams (2012) 29? Both 4 ? 8  62 69.1 ? 18.5 N/R N/R Carpenter (2012) 41? Both 3 mo ? 3y 781 66.0 ? 22.0 15.0 N/R Gordon (2008) Gordon (2008) 42? 42? Both Both 8 - 24 mo 8 - 24 mo 2472) 1333) 85.6 ? 30.7 89.9 ? 37.9 10.8 14.4 40.4 39.2 Kemp (2007) Kemp (2007) 40? 40? Both Both 1 ? 34) 1 ? 34) 78 78 84.4 ? 2.8 83.4 ? 3.0 N/R N/R N/R N/R Kemp (2007) Kemp (2007) 40? 40? Both Both 4 ? 85) 4 ? 85) 64 64 84.4 ? 2.8  63.2 ? 3.0 N/R N/R N/R N/R Stein et al (2006) 34? Girls 4 ? 8 168 93.8 ? 28.1 2.4 N/R 1)Data is presented as means and included SD where indicated 2) Infant group ? no specific definition of age 3) Toddler group ? no specific definition of age, 4)Summer, 5)Winter.  Chapter 2: Study 2.1 Purpose Despite the recognized importance of nutrition in young children and the known importance of vitamin D in calcium homeostasis and bone mineralization, knowledge of vitamin D intakes and status among young children is limited. The most recent DRIs for vitamin D issued by the U.S Institute of Medicine in 2011 for children 4 - 8 years of age were based on limited scientific data regarding vitamin D status in this age group. Although not addressed in this thesis, plasma 25 (OH)D that best reflect vitamin D deficiency,  insufficiency, and sufficiency in young children remain unclear, and to date no set cut-offs have been universally agreed upon by different expert groups. This study seeks to provide knowledge on dietary and supplemental intakes of vitamin D and the effects on plasma vitamin D status, based on measures of plasma 25 (OH)D in children aged 5 - 6 years of age living in Vancouver, British Columbia. Vancouver is located at latitude 49?N  and with an average of 60, 85, 134,182, 231 and 229 hours of sunshine for the months of January to June and 294, 268, 199, 125 64 and 56 h/month the months of July to December 34, and is a geographical area at which its inhabitants may be at risk for low capacity for endogenous vitamin D synthesis for much of the year. The purpose is to measure plasma 25 (OH)D concentrations, and assess concentrations in children compared to cut offs for deficiency, insufficiency, and sufficiency recommended in the 2011 DRI, and among children grouped by dietary intake of vitamin D, use of vitamin D supplements, and by season of the year.        31  2.2 Objectives The research in this thesis is designed with the following objectives for children 5 - 6 years of age living in Vancouver:  1. To estimate dietary vitamin D intake from natural and fortified food sources, and the intake of vitamin D from supplements 2. To determine the proportion of children meeting the EAR and RDA as set by the IOM (2011) based on their estimated vitamin D intake 3. To determine vitamin D status based on measures of plasma 25 (OH)D for whom dietary vitamin D has been estimated 4. To determine the proportion of children who, based on their plasma 25 (OH)D meet the criteria for vitamin D deficiency, insufficiency and sufficiency 5. To assess the importance of season of the year and vitamin D intake from foods and supplements in contributing to vitamin D deficiency and/or insufficiency when compared to vitamin D sufficiency   2.3 Methods 2.3.1 Design and setting This study is a cross-sectional study which was conducted at the University of British Columbia, Oak St Campus, at the Child and Family Research Institute.  Children and their parent or legal guardian (abbreviated as parents), all residents of greater Vancouver, were enrolled from the community between July 2010 and March 2013. Parents interested in participating with their child were asked to attend a research clinic, when their child was between 5 years 8 months and 5 years 11 months of age. Prior to collection of any information or test, the parent signed an informed consent form (Appendix A). Participants were assigned a randomly generated 4-digit  32  subject code and this was used on all data collection forms to maintain anonymity during the study. The range of ages over which children were seen was relatively narrow because of the potential for changes in developmental skills as well as food likes/dislikes with increased exposure to school, which cannot be adequately adjusted for by statistical approaches to control for differences in ages. Ethics approval for this research project was obtained from the University of British Columbia / Children?s and Women?s Health Centre of British Columbia Research Ethics Board (UBC C&W REB) a UBC-affiliated Research Ethics Board (REB) for the Oak Street campus. In addition, ethical approval to approach and recruit subjects in the community was obtained from Vancouver Coastal Health, and Vancouver School Board Research Ethics Committee.   2.3.2 Inclusion and exclusion criteria and recruitment Inclusion criteria were that the parent (usually the mother) was comfortable speaking and writing English language, and that the child was born after full-term gestation (>37 wk gestation) with no congenital or acquired disease considered likely to impact healthy child growth and development. The children had to be between the ages of 5 years 8 months and 5 years 11 months of age at the time when they participated in the study. Children with iron deficiency were to be withdrawn from the study, based on hematocrit levels below 34.  Exclusion criteria were all children not meeting the inclusion criteria.     2.3.3  Demographic characteristics Information on each child?s family background was collected using confidential questionnaires labeled only with the child?s random number code. Information on the mother?s age, ethnic background, highest level of education, number of adults and children in the  33  household, smoking and other relevant information was collected. Total annual family income was recorded as $20,000 (or less) to $80,000 (or more) in $10,000 increments (Appendix B).      2.3.4 Dietary assessments and collection of information on supplement use To assess each child?s dietary intake, both a Food Frequency Questionnaire (FFQ) and three 24-hour dietary recalls were conducted. The FFQ interview and first 24 hour dietary recall were conducted by myself or another trained interviewer during the study visit. The 24 hour recall was done with food models, cups, spoons and packages to assist the parent in estimating portion sizes. A multiple step approach (5 pass) was used for the 24 hour recalls 57,58. Briefly, the participant was asked to list uninterruptedly, all foods and beverages consumed by the child during the previous 24 hours. Next, the participant was asked about common forgotten foods, such as condiments, drinks and snacks. The participant was next asked for time, location and occasion when the foods and beverages were consumed. Using the food models, the parent was then asked for descriptions and quantities of foods and beverages consumed. Following this, the list was reviewed to ensure no foods had been forgotten. Finally, the participant was asked if the child had been given any supplements. If so, brand and amount of supplements given were recorded on the bottom of the page. If no supplements had been given ?No supplements? was recorded to indicate that the participants had been asked this. A second and third unscheduled 24- hour recall was conducted by telephone using the exact same procedure. Each parent was asked for the most convenient time to call. The random telephone calls were conducted over the following two weeks to capture a total of one weekend day and two week days of records for each child.  The FFQ was interviewer-administered, and utilized to collect details on the frequency and portions sizes of foods eaten over the past 4 weeks. The FFQ was used to capture details on the intake of foods, in particular major protein sources, such as fish and meats that are not eaten  34  on a daily basis and for which single or 3 day records may therefore result in errors in estimating average intakes for the individual. The FFQ contains 14 main food categories that include different food items, with sub-categories for fat content; for example whole, 2 %, 1 % or skim milk. Another example is the specific types of fats, such as margarine and butter, yogurts, cream cheeses, types of vegetable oils, salad dressings and mayonnaise, fresh, frozen and canned fish, and types of fish and shell fish, as well as eggs, meats and poultry. The FFQ collects information on both the amount and frequency of consumption, and allows for addition of items not on the list, or other portion sizes (Appendix C).  Further, information on supplement use, including brands and when the use of these commenced was collected with the FFQ.   2.3.5 Analysis of dietary intakes The dietary information was entered into nutrient analysis software (ESHA Food Processor SQL. Version 10.10.0.0, Salem, OR: ESHA Research, 2012), to enable analysis of each child?s intake of energy and all nutrients, and to create food lists with the amounts of different nutrients from different foods. The nutrient software contains the Canadian Nutrient File (CNF) 19 and United States Department of Agriculture (USDA) nutrient data base for several thousand foods and specific food brands. Home prepared foods and restaurant foods, as needed, were disaggregated for entry into the database. The FFQ and 24 hour recalls were entered by myself or another research assistant, and vitamin D intakes retrieved from the database as ?g/day. All data was cross-checked to ensure accuracy and consistency of the data entry. Dietary vitamin D intake was calculated using both the FFQ and three 24 hr recalls and intakes were calculated as the average intake in ?g/day and intake/1,000 calories/day. Supplement data was calculated using both the FFQ and three 24 hour recalls, and intakes  35  calculated as the average intake/day. When the supplement brand or amounts of supplements given was not recorded or known by the participant a standard amount of 10 ?g vitamin D was assumed or the intake recommendations of the manufacturer of a specific brand used when type but not dose was known. Incomplete data was entered as missing values.    2.3.6 Anthropometrics For each child, standing height and weight was measured using standardized, calibrated equipment in the Clinical Research Unit at the Child and Family Research Institute. Height and weight were each measured twice and reported as an average of the two. Z-scores for height-for-age, weight-for-age and BMI-for- age were calculated using the World Health Organization?s (WHO) AnthroPlus software 59.    2.3.7 Blood collection, preparation and analysis of 25(OH)D Venous blood was collected by a registered technician following completion of all other assessments, and data collection with the child and parent. Directly after collection, the blood was transferred to the laboratory, then immediately centrifuged at 3700 rpm for 10 min, 4?C to separate the plasma and blood cells. The plasma was aliquoted into storage tubes, sealed, labeled and stored at -80 ?C until analysis.   2.3.7.1 LC-MS/MS Plasma concentrations of (25OH)D were used as a biochemical measure for vitamin D status using LC- tandem mass spectrometry (MS/MS). The LC-MS/MS is a Waters ACQUITY UHPLC system connected to a Quattro micro triple quadrupole mass spectrometer (Waters Canada, Mississauga, Ontario). Briefly, deuterium-labeled 25-hydroxy vitamin D3-d6 Calcidiol  36  (26,26,26,27,27,27-d6) (Chemphor, Canada, Ottawa, Ontario # CHE011) in 15 ?l was added to 75 ?L plasma. Proteins were precipitated with 1 volume of acetonitrile, the mixture vortexed for 10 seconds, then left at room temperature for 5 minutes.  Thereafter, the mixture was vortexed again for 5 - 10 seconds, and then centrifuged at 15,000g for 10 minutes to pellet the proteins and the supernatant was recovered. Next, 400 ?l ammonium formate (in 0.01 M water)  was added, the sample vortexed 5-10 seconds, 1ml of ethyl acetate, added the tube vortexed for 10 seconds and then left at room temperature for 2 - 3 minutes. After vortexing again for 5 - 10 seconds, the sample was centrifuged at 2000 rpm for 5 minutes to obtain well defined aqueous and organic layers. The top layer (ethyl acetate) was removed to brown tinted autovials and dried under nitrogen. An additional 1ml of ethyl acetate was added to the remaining lower layer to re-extract and recover any 25 (OH)D remaining, and this was pooled with the first extract. The samples were then dried under nitrogen, 75?l of the derivatizing agent, 1 mg/ml 4-phenyl-1,2,4-triazonile-3,5-dione (Sigma-Aldrich, Canada, Oakville, Ontario,  # 280992) in acetonitrile (PTAD/acetonitrile) was added and the vials left in the dark for 1 hour at room temperature. Then, 55 ?l ammonium formate (in 0.01 M water) was added and the samples were transferred to 150 ?l autosampler inserts each with a clean plastic transfer pipette. Chromatography was accomplished on a ACQUITY UPLC BEH C8 column, 1.7?m, 2.1mm x 50 mm (Waters Canada, Mississauga, Ontario) with a binary mobile phase gradient which were, ammonium formate in water (A) and methanol (B), both containing 0.1 % formic acid (v/v), commencing at 85 % (A) 15 % (B) and changing to 30 % (A) and 70 % (B). The column was flushed with 100 % methanol before returning to initial conditions. The sample injection volume was 3 ?L and accomplished with an auto-sampler and temperature-controlled sample chamber held at 5oC. A standard curve was created using standard solutions of 5, 10, 20, 40, 60, 90, 120, 150 and 200 ng/mL. Quality control samples were made using internal standard (15ul) plus 75?l of methanol,  37  with a reagent blank made using 90 ?l methanol were analyzed with each sample batch. The intra assay CV was 3.6 % and the inter-assay was 8.7 %. In method development, serum samples in which 25 (OH)D had been analyzed by radio- immunoassay were generously provided by Dr. Tim Green, University of British Columbia, and these were analyzed to compare the results as analyzed by LC-MS/MS. The correlation coefficient between the RIA and UHPLC analyses was r= 0.88.   2.3.8 Statistical analysis Statistical analyses were performed using IBM SPSS statistics version SPSS software (IBM SPSS Statistics for Windows, Version 21.0, 2012. Armonk, NY: IBM Corp). All dietary data was checked for normality using the Kolmogorov-Smirnov?s test of normality, with a significance level < 0.05 used to indicate a deviation from the normal distribution. Descriptive statistics, including, percentages, means ? SD, medians and inter quartile ranges (IQR), and 2.5th ? 97.5th percentile were used to summarize the data collected. Subsequently, the statistical analyses were based on means and medians using parametric and non-parametric tests as applicable. Pearson correlation was used to determine the correlation within individuals between energy intake as well as vitamin D intake when assessed using the FFQ compared to a single 24 hr recall, or the average of three 24 hr recalls. The proportion of dietary vitamin D obtained from different foods (contribution of major dietary sources to vitamin D intake) was calculated for each child using data from the FFQ. Additionally, the proportion of vitamin D derived from natural, fortified foods and supplements were calculated for each child, then the group means and medians calculated. Children were grouped based on vitamin D status as sufficient (? 50 nmol/L), insufficient (50 ? 30 nmol/L) and deficient (< 30 nmol/L) based on the cut-off values defined by the IOM (2011) 3.The proportion of children who were deficient, insufficient and sufficient during different seasons was also determined.  The FFQ and three 24 hr recalls were  38  used to quantify the proportion of children meeting the EAR and RDA for vitamin D based on diet alone, as well as diet and supplements in combination. Further, children were split into supplement users and non-supplement users, and the proportion of children meeting the EAR and RDA for each group determined for the two dietary data collection methods was quantified. Spearman correlation was used to determine whether there was a correlation between vitamin D intake and plasma 25 (OH)D. A P value  ? 0.05 was considered significant.                    39  Chapter 3: Results Figure 3.1 Study flow chart                          1 Excluded, n= 1 did not meet inclusion criteria (second of a twin pair); 2 demographic data excluded, n= 5 (they were fathers); missing data for ethnicity and household income, n=1; excluded due to incomplete data, n=3; 4 excluded sick child or incomplete data, n= 5; 5 lost to follow-up or unreliable data for 24 hr recalls, n=9; 6supplement data n= 81 assumed manufacturer recommended dose, and n =46 assumed content (10 ?g for MVI).   Consented, n=200 Research study appointment1  Supplement data FFQ, n=1936 Supplement data 3 x 24 hr recall, n=181   Child assessment  Parental interviews  FFQ, n=1963  Anthropometrics, n=199  Blood sample, n=154 n Demographics, n=1942  1 x 24 hr recall, n= 1944  3 x 24 hr recall, n= 1835   40  3.1 Subject characteristics   From July 2010 to March 2013, 200 children who met all inclusion criteria were enrolled in the study. However, one twin pair was enrolled, and therefore the second child of the twin pair was excluded, as per protocol. No child was found to have a low hematocrit, thus none were excluded on the basis of concerns over this measure of iron status. Of the children, 48.2 % were boys and 51.8 % were girls. The children?s age was controlled by the study inclusion criteria, and thus showed little variation, with a mean age of 68.7 ? 0.7 months. Of the parents who attended the research study, 194 were mothers and five were fathers. Information on household income was reported by n= 193 parents of whom a large proportion reported an annual household income above $50,000 CAD (85.0 %). An income between $30,000 to $50,000 CAD was reported by 9.8 % of parents and 5.2 % reported having an annual household income below $30,000 CAD. Information on ethnic background for both mother and father were recorded, but only information for the mother is presented. Information on the mothers? ethnicity was available for n= 193, of whom 70.0 % were of Caucasian background, 14.0 % were Chinese, and 3.6% were of East Indian background. Further, 3.6 % were of other Asian backgrounds, and 9.3 % were grouped as from other backgrounds as their mothers identified themselves having a mix of ethnicities. No individuals of African American background and only one individual of First Nations descent (corresponding to 0.5 %) were enrolled in the study, with the latter child grouped under the ?others? category.  In addition, of the parents from whom information on education was available (n=194), 28.9 % reported having a university graduate degree, and 39.7 % reported having a university undergraduate degree. Further, 26.8 % reported having a college degree or a diploma and 4.6 % reported having a high school diploma or less. Maternal age at the research study visit was divided into four groups; 2.6 % reported being in the age category 20 ?  41  29 years, 43.8 % were between 30 ? 39 years, 53.6 % were 40 ? > 49 years, with one woman (0.5 %) being over fifty years (Table 3.1).                         42  Table 3.1 Subject characteristics         Characteristic N  Sex of children (%), boys / girls 199 48.2 / 51.8 Age in months at time of visit (Mean ? SD) 199 68.7 ? 0.7 No. of children in home  (Mean ? SD) 198 2.0 ? 0.6 No. of individuals in home (Mean ? SD) 198 4.1 ? 0.9 Household income level (%) 193  < $30,000  5.2 $30,000 - $50,000  9.8 > $50,000  85.0 Mother?s ethnicity (%) 193  White/Caucasian  70.0 Chinese  14.0 Other  9.8 East Indian  3.6 Other Asian  3.6 Mother?s highest education level (%) 194  High school or less  4.6 College/diploma  26.8 University undergraduate degree  39.7 University graduate degree  28.9 Mother?s age (%) 194  20 ? 29 years  2.6 30 ? 39 years  43.8 40 - > 49 years  53. 6 Of the children included in the study n= 96 were boys and n=103 were girls.   43  Based on the World Health Organization?s (WHO) reference curves59, the mean ? SD z-score for weight-for-age was 0.18 ? 0.95, and 75.4 % had weight-for-age within  -2 SD to +2 SD, 7 % were classified as low weight-for-age (<-2 SD) and 17.6 % had a high weight for-age (> +2 SD, including one child > +3 SD). The mean height-for-age z-score was 0.17 ? 0.97, and 70.4 % were within the normal range, 9.0 % had a low height-for-age and 20.6 % had a height-for- age z-score > +2SD. The mean BMI-for-age was 0.09 ? 0.99, 9.5 % had a low BMI z-score <-2 SD and 14.6 % had a BMI z-score > +2 SD, and 75.9 % had a BMI-for-age between -2 SD to +2 SD. Only one child had a BMI-for-age z-scores (and weight-for-age z-score) > +3 SD and this child was included in the previous category (Table 3.2).   Table 3.2 Child anthropometric measures    Weigh-for-age Z-score     0.18 ? 0.95 Height-for-age Z-score   0.17 ? 0.97 BMI-for-age Z-score  0.09 ? 0.99        Low BMI-for-age (%)   9.5        Normal BMI-for-age (%)  75.9        High BMI-for-age (%)  14.6 Results are as means ? SD, % as indicated, n=199. Low BMI-for-age was defined as the percentage of children with a BMI Z-score <-2 SD, normal BMI-for-age was defined as BMI Z-score between- 2 SD and + 2 SD and high BMI-for-age was defined as BMI Z-score > +2 SD, based on WHO?s growth curves. Only one child had BMI-for-age >3+SD as was included in the high BMI-for-age category.    44  3.2 Vitamin D intake  Dietary vitamin D intake data was skewed to the right, with a higher mean than median when estimated using both the FFQ and 24 hr recalls. Analysis of the FFQ, the single 24 hr recall for the day preceding the study visit, and the average of three 24 hr recalls all showed that boys had a higher energy intake than girls. Boys and girls, respectively, had median energy intakes of 1833 (IQR 786) kcal/day and 1690 (IQR 545) (p= 0.019) estimated using the FFQ, 1651(IQR 612) kcal/day and 1344 (IQR 548) kcal/day (p< 0.001), respectively, using a single 24 hr recall, and 1577 (IQR 452) kcal/day and 1388 (395)kcal/day, respectively, using the three 24 hr recalls (p<0.001) (Table 3.3).   Table 3.3 Energy intakes (kcal/day) estimated using FFQ and 24 hour dietary recalls   FFQ n =196 One 24 hr recall n =194 Three 24 hr recall n = 183 Mean ? SD   1840 ? 492 1548 ? 438 1517 ? 331 Median (IQR) 1771 (688) 1498 (655) 1497 (429) 2.5th ? 97.5th 1034 ? 3034 848 - 2558 927 ? 2231     Boys median (IQR) 1833 (786) 1651 (612) 1577 (452)     Girls Median (IQR)  1690 (545)* 1344 (548)* 1388 (395)* Children included were n= 94, 94 and 87 boys and 102, 100 and 96 girls for results for the FFQ, single 24 hr recall and three 24 hr recall, respectively. * Values girls significantly different from boys, P=0.019, P< 0.001, P=<0.001 for the FFQ, single 24 hr recall and three 24 hr recalls, respectively.     45  Figure 3.2 - Panels A and B, show energy intake for each child estimated from the FFQ plotted against energy intake for the same child estimated using a single 24 hr recall (n=192) or the three 24 hr recalls (n=182). The correlation coefficients for the estimated energy intake using the FFQ compared to one 24 hr recall or the three 24 hr recall were r = 0.403 (p< 0.001), r = 0.458 (p< 0.001), respectively (Figure 3.2).   Figure 3.2 Inter-individual correlation between energy intake estimated using the FFQ and a single 24 hr recalls, and between energy intake estimated using the FFQ and three 24 hr recalls  A)       B)     The plots show the correlation and 95 % CI of the regression line. Panel A, FFQ and single 24 hr recall; Panel B, FFQ and three 24hr recalls.    Dietary vitamin D intakes based on the FFQ (n=196), the single 24 hr recall (n=194) and the three 24 hr recalls (n=183) for all children and for the boys and girls separately are shown in Table 3.4. The median vitamin D intake for all children was 6.3?g/day (IQR 4.6), 4.2?g/day (IQR 4.6) and 4.7?g/day (IQR 3.5) when estimated using the FFQ, single 24 hr recall and three 24 hr recall, respectively. Thus, the median vitamin D intake was 33.3 % and 25.4 % higher  Caloric intake three 24 hr recalls  Caloric intake single 24 hr recall Caloric intake FFQ Caloric intake FFQ 500 500 500 1500 1500 1500 1500 1000 1000 2000 1000 2000 2000 2000 2500 2500 2500 2500 3000 3000  3000 3000       n =192 r = 0.403 p<0.001 n =182 r =0.484 p<0.001 1000 500   46  when estimated using the FFQ than when estimated using the single 24 hr recall (P<0.001) or the three 24 hr recalls (P<0.001), respectively. The difference in the median vitamin D intake estimated using a single 24 hr recall or three 24 hr recalls was not significantly different (P= 0.412). Vitamin D intake estimated with the FFQ was significantly correlated with intake estimated using a single 24 hr recall (r= 0.538, P<0.001), or three 24 hr recalls (r= 0.500, P<0.001), respectively. Boys had a significantly higher median vitamin D intake than girls across all the three dietary methods (Table 3.4), although the difference in vitamin D intake between boys and girls was statistically significant when assessed using the FFQ (P= 0.015), but not the single 24 hr recalls (P= 0.192) hr recall, with a strong trend for the three 24 hr recalls (P=0.052). To assess whether the higher vitamin D intake estimated using the FFQ was caused by higher food intakes, vitamin D intakes were also analyzed per 1000 kcal. Adjusting for energy intake decreased the difference in median vitamin D intakes for all children obtained by FFQ from 33.3 % to 16.2 % when compared to the single 24 hr recalls, and from 25.4 % to 13.5 % for the three 24 hr recalls. However, the difference in median vitamin D intake was still significantly higher when estimated using the FFQ than a single recall (P=0.001), or three 24 hr recalls (P=0.006). The median vitamin D intake per 1000 kcal was not significantly different when estimated using the single and three 24 hr recalls (P= 0.157). The median vitamin D intake was also not significantly different between boys and girls when estimated using the FFQ (P=0.198), single 24 hr recall (p=0.473) or three 24 hr recalls (p=0.338).        47  Table 3.4 Daily dietary vitamin D intake and vitamin D intake as ?g/1000 kcal from foods estimated using the FFQ, single 24 hr recall and three 24 hr recalls.   FFQ n= 196 One 24 hr recall n= 194 Three 24 hr recall n=183 Vitamin D ?g/day    Mean ? SD 6.7 ? 3.7 5.1 ? 3.8 5.3 ? 3.5 Median  (IQR) 6.3 (4.6)* 4.2 (4.6) 4.7 (3.5) 2.5th ? 97.5th 1.7 ? 16.8 0.4 ? 16.3 0.9 ? 13.5 Boys  median (IQR) 7.1 (5.6) 4.5 (5.1) 5.3 (4.2) Girls median (IQR) 5.8 (4.2) 4.1 (4.2) 4.4 (3.1) Vitamin D  ?g/1000 kcal    Mean ? SD 3.7 ? 1.7 3.4 ? 2.5 3.5 ? 2.1 Median  (IQR) 3.7 (2.3)+ 3.1 (2.9) 3.2 (2.5) 2.5th ? 97.5th 0.8 ? 7.6 0.2 ? 11.1 0.5 ? 9.8 Boys  median (IQR) 3.9 (2.4) 3.3 (2.7) 3.3 (2.5) Girls median (IQR) 3.6 (2.2) 2.8 (2.9) 3.0 (2.5) FFQs were analyzed for 94 boys and 102 girls, a single 24 hr recalls were analyzed for 94 boys and 100 girls, and three 24 hr recalls were analyzed for 87 boys and 96 girls. * Intake significantly higher for FFQ than one or three 24 hr recalls (P<0.001). + Intake significantly higher for FFQ than one 24 hr recall (P=0.001) or three 24 hr recalls (P=0.006).    48  3.2.1 Vitamin D intakes from foods including natural sources and fortified foods  Table 3.5 shows vitamin D intake from different foods as ?g/day estimated using dietary information collected by the FFQ. The amount of vitamin D obtained from each food group was calculated individually for each child, after which the group mean was calculated. The major dietary source of vitamin D was fortified milk and milk alternatives, and these combined contributed 62.6 % of daily dietary vitamin D intake, corresponding to a mean ? SD and median intake of 4.2 ? 3.1?g/day and 3.7?g/day, respectively. Fish provided 19.4 % of the daily dietary vitamin D intake. Other dairy products, eggs and margarine each provided 3 % of the daily dietary vitamin D intake. Other food sources together contributed 9.0 % of the dietary vitamin D, and these included meat and combination meals for which individual ingredients could not be put into specific categories (Table 3.5).   Table 3.5 Dietary vitamin D intake (?g/day) and proportion of total intake obtained from different foods Food  Mean ? SD (?g/day) Median (IQR) (?g/day) 2.5th ? 97.5th (?g/day) % total intake Fluid milk 4.2 ? 3.1 3.7 (4.0) 0.5 ? 12.3 62.6 Other dairy 0.2 ? 0.2 0.2 (0.2) 0.0 ? 0.8 3.0 Margarine 0.2 ? 0.5 0.0 (0.2) 0.0 - 2.0 3.0 Fish  1.3 ? 1.6 0.6 (2.1) 0.0 ? 5.4 19.4 Eggs  0.2 ? 0.2 0.2 (0.3) 0.0 ? 0.7 3.0 Other sources   0.6 ? 0.5 0.6 (0.4) 0.1 - 1.5 9.0  Total 6.7 ? 3.7 6.3 (4.6) 1.7 ? 16.8 100 FFQ were analyzed for 196 children. Results are for foods only do not include supplements.  Percentages are determined from the mean intakes.   49  3.2.2 Vitamin D intakes from supplements Information on whether or not the child was given vitamin D containing supplements was collected using two different methods. First, information on whether a supplement was given was collected and the child was classified as a supplement user if they had been given a vitamin D containing supplement on at least one of the three 24 hr recall days.  Second, a question included in the FFQ asked the amount and how often supplements were given within the last month. For the purpose of this analysis, children who based on the FFQ question had been given a vitamin D containing supplement at least twice a week were considered as supplement users.  Based on the FFQ, 70.5 % of the children were given supplements at least twice a week. In contrast, only 57.7 % of the children were given a supplement on at least one of the three days of the diet recalls. The vitamin D intake obtained from supplements was skewed, regardless of whether or not supplemental vitamin D intake was estimated by the FFQ or from the three 24 hr recalls. Children who were given vitamin D containing supplements had a median intake of vitamin D from supplements of 10.0 ?g/day (IQR 7.3 ?g/day) estimated using the FFQ, with no significant difference in the median intake from supplements between boys, (10.0 ?g/day, IQR 6.4?g/day, n= 68) and girls (10.0 ?g/day, IQR 5.0?g/day, n= 68). The median vitamin D intake by in children from supplements who reportedly were given supplements based on the three 24 hr recalls was 8.3 ?g/day (IQR 8.2?g/day), with a median intake of 10.0 ?g/day (IQR 7.5?g/day) among boys (n=51) and 6.7?g/day (IQR 6.7 ?g/day) among girls (n= 57), with the difference in intake between boys and girls of borderline significance (P= 0.057) (Table 3.6).         50  Table 3.6 Vitamin D intake from supplements, ?g/day  FFQ   Three 24hr recall    Mean ? SD  11.0 ? 9.3 10.0 ? 7.9  Median (IQR) 10.0 (7.3) 8.3 (8.2)  2.5th ? 97.5th 0.7 ? 44.7 3.0 ? 38.0     Boys median (IQR)  10.0 (6.4) 10.0 (7.5)      Girls median (IQR) 10.0 (5.0) 6.7 (6.7)* 1Data includes children who were given supplements only, the FFQ included 68 boys and 68 girls, and the three 24 hr recalls included 51 boys and 57 girls. *P= 0.057 compared to respective value for boys. No significant differences between estimates based on the FFQ and three 24 hr recalls were found  3.2.3 Vitamin D intake from foods and supplements Next, we assessed whether vitamin D intakes from foods were different between children given vitamin D containing supplements and children not given supplements. No significant differences in dietary vitamin D intake from foods was found between children given and not given supplements (P >0.05), regardless of the dietary methodology (Table 3.7).            51  Table 3.7 Vitamin D intake as ?g/day from foods in children given or not given vitamin D containing supplements  FFQ Three 24 hr recalls   Supplements n=134 No supplements n=56  Supplements       n=103 No supplements n=77 Mean, SD  6.6 ? 3.8 6.8 ? 3.5 5.2 ? 3.5 5.4 ? 3.7 Median (IQR) 6.1 (4.6) 6.8 (5.0) 4.6 (3.3) 4.7 (3.6) 2.5th ? 97.5th   1.6 ? 17.1 1.6 ? 14.4 0.6 ? 15.5 1.3 ? 14.3 Values are ?g/day and represent only children who provided information on both dietary and supplement intake.  Table 3.8 shows the total daily vitamin D intake from food plus supplements for children given supplements and children not given supplements. This data shows the extent to which supplements increase the total vitamin D intake in children given supplements compared to children not given supplements. Data derived from the FFQ showed that children given supplements had a median (IQR) intake of 16.1 ?g/day (IQR 9.4), which is more than two fold higher than the median (IQR) vitamin D intake of 6.8 ?g/day (IQR 4.9) in children not given supplements. Similarly, using data derived from the three 24 hr recalls, children given supplements had a median (IQR) of 13.0 ?g/day (IQR 9.0), also more than two fold higher than the median intake of  4.8 ?g/day (3.7) for children not given supplements (Table 3.8).         52  Table 3.8 Total vitamin D intake from diet and supplements or diet alone estimated using the FFQ and three 24 hr recalls for children given or not given supplements1.           Supplemental vitamin D   Yes No All  children FFQ, ?g/day n = 134 n= 56 n= 190 Mean ? SD 17.2 ? 9.5 6.8 ? 3.4 14.2 ? 9.4     Median (IQR) 16.1 (9.4)* 6.8 (4.9) 12.4 (10.9)          2.5th ? 97.5th 3.8 ? 45.1 1.6 ? 14.4 2.0 ? 40.9     Three 24 hr recalls ?g/d n= 104 n= 76 n=180 Mean ? SD 15.1 ? 8.6 5.4 ? 3.7 11.1 ? 8.5     Median (IQR) 13.0 (9.0)* 4.8 (3.7) 9.1 (8.5)          2.5th ? 97.5th 5.4 ? 42.9 1.3 ? 14.6 1.4 ? 35.5 1Data includes only children for whom data on both diet and supplements was available. * Intake significantly higher than in children not given supplements, P<0.001.  3.2.4 Vitamin D intakes compared to the EAR and RDA for children The proportion of vitamin D obtained from supplements, and the intakes from fortified foods and natural food sources was estimated for each child using data obtained with the FFQ. The mean vitamin D intake from foods and supplements for all children was 14.2 ? 9.4 ?g/day. Of this, 52.8 % of the total mean vitamin D intake was from supplements, representing a mean of 7.5 ? 8.7?g/day. Natural foods provided 14.1 % of the total vitamin D intakes, equivalent to a mean of 2.0 ? 1.7?g/day, and 33.1 % of the total vitamin D intake was provided by fortified foods. Notably, 85.9 % of total vitamin D intakes were from fortified foods and supplements. The mean vitamin D intake from diet plus supplements of 14.2 ?g/day for all children exceeds  53  the EAR of 10?g/day. However, the EAR would not be reached if children were not given supplements, i.e. the mean intake from natural foods and fortified foods alone is below 10 ?g/day in children. Further, the intake would be substantially less if vitamin D food fortification was not in place, as the mean intake from natural foods was only 2.0 ? 1.7 ?g/day (Table 3.9, Figure 3.3).  Table 3.9 Proportion of total daily vitamin D obtained from supplements, fortified food and natural food sources assessed by FFQ    Mean ? SD Median (IQR) Total (%) Supplements FFQ  7.5 ? 8.7 6.8 (10.0) 52.8  Dietary intake   6.7 ? 3.7 6.3 (4.6) 47.2        Natural food sources  2.0 ? 1.7 1.4 (2.0) 14.1        Fortified food sources   4.7 ? 3.1 4.0 (3.9) 33.1  Fortified foods and supplements  12.2 ? 9.0 10.8 (11.1) 85.9  Total intake food and supplements  14.2 ? 9.4 12.4 (10.9) 100  n=190. Data were analyzed only for children for whom information on both diet and supplement intake were provided, and includes children given no supplements. Percentages calculated using means.        54  Figure 3.3 Amount of vitamin D consumed in natural food sources, fortified foods and supplements with comparison to the EAR and RDA      Data is derived from the FFQ and was analyzed for 190 children who provided information on both dietary and supplement intake of vitamin D. Percentages based on mean intakes. The EAR and RDA are 10 ?g/day and 15 ?g/day16.   Of the children, 82.7 % and 90.7 % did not meet the EAR for vitamin D intake from dietary sources when estimated using the FFQ or three 24 hr recalls, respectively. However, the proportion of children not meeting the EAR decreased to 34.7 % and 51.4 % respectively, when vitamin D intake from supplements was included (Figure 3.4 A). The proportion of children not meeting the RDA based on diet alone was 96.4 % and 98.4 % for the FFQ and three 24 hr recalls, respectively (Figure 3.4 B). Combining dietary and supplemental sources of vitamin D, the proportion of children not meeting the RDA was 57.4 % and 76.0 % for the two dietary methods, respectively (Figure 3.4 B).  1 3 5 7 9 11 13 15 Diet + supplements Supplements Diet  Fortified foods Natural foods RDA EAR Vitamin D intake ?g/day Vitamin D sources  55  Figure 3.4 Proportion of children with intakes of vitamin D from diet or diet plus supplements with vitamin D intakes below the EAR or RDA for vitamin D    Proportion of children meeting or not meeting the EAR (?10 ?g/day) (panel A) or RDA (?15 ?g/day) for vitamin D (panel B). The data is based on intake from the FFQ from diet (n=196) or diet and supplements (n=190) and intake from the three 24 hr recalls from diet (n=183) or diet and supplements (n=179).   56  To illustrate the potential importance of supplement use, the children were divided into supplement users and non-supplement users who met or did not meet the EAR (?10 ?g/day) or RDA (15 ?g/day) for vitamin D. Of the supplement users, as few as 15.7 % and 24.0 % of the children did not meet the EAR, whereas 80.5 % and 89.5 % of the non-supplement users did not meet the EAR based on analysis of the FFQ and three 24 hr recalls, respectively (Figure 3.5 A). Of the supplement users, 42.5 % and 59.6 % did not meet the RDA, based on the two dietary data collection methods, respectively. Conversely, 92.9 % and 98.7 % of children not given supplements failed to meet the RDA for vitamin D, based on the two methods, respectively (Figure 3.5 B).                 57  Figure 3.5 Proportion of children given or not given vitamin D supplements with a total intake of vitamin D below or above the EAR or RDA        Proportion of children given or not given supplements consuming below or above the EAR (10?g/d) (Panel A) or RDA (15?g/d) (Panel B) for vitamin D. Results are based on data collected by the FFQ for supplement users (n= 134) and non supplement users (n= 55) and three 24 hr recalls for supplement users (n=103) and non-supplement users (n= 76). Data only includes dietary data from children who provided a blood sample.   58  3.3 Plasma 25 (OH)D Plasma 25 (OH)D was analyzed for 154 children who provided blood, of these 71 were boys and 83 were girls. The mean ? SD 25 (OH)D for all children was 64.4 ? 17.3 nmol/L. The plasma 25 (OH)D was normally distributed in the whole group and in the boys (P= 0.074) and girls (P= 0.2). Boys and girls also had similar 25 (OH)D concentrations with a mean ? SD of 65.5 ? 16.9 nmol/L for boys and 63.5 ? 17.6 nmol/L for girls (P=0.473). The plasma 25 (OH)D was grouped for children given supplements (n= 104) and not given supplements  (n=46). The mean ? SD 25 (OH)D for children given supplements was 66.4 ? 16.2 nmol/L and 58.8 ? 18.4 nmol/L for children not given supplements (Table 3.10). Although the plasma 25 (OH)D for supplement users was normally distributed (P= 0.200) in children given supplements, it was skewed in children not given supplements (median IQR of 54.4, 25.3 ?g/day),  P= 0.012 with a lower median than mean value. Analysis by Mann-Whitney U showed that plasma 25 (OH)D was significantly different between children given (median 65.0, IQR 22.2 ?g/day) and not given supplements (P= 0.006), corresponding to 16.3 % higher median 25(OH)D in children given supplements.    Table 3.10 Effect of supplement use and sex on plasma 25 (OH)D in children.  Mean ? SD  Median (IQR) 2.5th ? 97.5th   All, n=154 64.4 ? 17.3 62.2 (24.0) 31.5 ? 105.4 Boys, n= 71 65.5 ? 16.9 61.7 (23.9) 32.9 ? 111.7 Girls, n= 83 63.5 ? 17.6 62.4 (25.7) 20.9 ? 103.7 Supplements, n= 104 66.4 ? 16.2 65.0 (22.2) 38.5 ? 106.9 No supplement use, n= 46 58.8 ? 18.4 54.4 (25.3)*+ 21.1 ? 112.3 Data is presented as nmol/L. *Data is skewed P= 0.0012, +significantly different from children given supplements P=0.006.  59  3.3.1 Plasma 25 (OH)D and vitamin D intake of children during different seasons Vitamin D intake across four different seasons was analyzed to estimate whether vitamin D intake differed during the year. The year was divided in to four seasons where winter was defined as January to March, spring as April to June, summer as July to September and fall as October to December. The number of children in each of the four seasons with complete FFQ was 59, 42, 43 and 46, respectively, and 55, 39, 39 and 47 for three 24 hr recalls, respectively. The distribution of vitamin D intakes were not normally distributed within any season. However, there were no significant differences in vitamin D intake from foods and supplements across seasons when estimated using the FFQ (P= 0.28) or three 24 hr recalls (P= 0.199), thus higher vitamin D intakes during a specific season is not expected to explain any seasonal differences in plasma 25 (OH)D (Table 3.11).  Table 3.11 Vitamin D intake from diet and supplements during different seasons  Winter Spring Summer Fall P value  FFQ n= 59 n= 42 n= 43 n= 46  Mean ? SD 15.3 ? 9.6 15.3 ? 10.7 13.0 ? 6.5 12.9 ? 10.3            Median 15.1 (13.1) 13.7 (9.3) 12.1 (8.8) 10.5 (12.5) 0.28  Three 24 hr recalls  n= 54 n= 39 n= 39 n= 47   Mean ? SD 12.9 ? 8.7 10.4 ? 8.1 8.6 ? 4.0 11.5 ? 10.7            Median 11.7 (12.4) 9.0 (8.4) 8.1 (5.0) 7.9 (12.6) 0.199 Data are ?g/day and includes children who provided information on both diet and supplements only. Winter, spring, summer and fall was defined as January to March, April to June, July to September and October to December, respectively.   60  Of the 154 children who provided blood, 81.2 % were classified as vitamin D sufficient, 16.9 % insufficient and 1.9 % deficient, based on the cut-off of <50 nmol/L and < 30 nmol/L for insufficiency and deficiency, respectively, defined by the IOM 3. Fifty three (n= 53) children were seen during winter months (January - March) and 75.4 % of these children were classified as sufficient, 20.8 % insufficient and 3.8 % deficient. During spring (April ? June), 32 children provided a blood sample and of these 87.5 % were classified as sufficient, 12.5 % insufficient and no children were vitamin D deficient. Thirty two (n= 32) children was seen during summer (July ? September), and of these children 93.6 % were classified as sufficient, 3.2 % were insufficient and 3.2 % (one child) were deficient. Fall was defined as October ? December and 38 children were seen during this season, with 73.7 % classified as sufficient, 26.3 % insufficient, and no children were classified as deficient (Table 3.12).   Table 3.12 Percentage of children classified as vitamin D sufficient, insufficient and deficient, year round and during different seasons  ? 50 nmol/L 30 ? 50 nmol/L ? 30 nmol/L Year round, n= 154 81.2 (125)1 16.9 (26) 1.9 (3) Winter, n= 53 75.4 (40) 20.8 (11) 3.8 (2) Spring, n= 32 87.5 (28) 12.5 (4) 0.0 (0) Summer, n= 31 93.6 (29) 3.2 (1) 3.2 (1) Fall, n= 38 73.7(28) 26.3 (10) 0.0 (0) Winter, spring, summer and fall was defined as January to March, April to June, July to September and October to December, respectively.1The brackets are (n) children classified within the 25 (OH)D cut-off value.    61  Overall, for the entire study, only three children were classified as vitamin D deficient, with 25 (OH)D values of 19.2 nmol/L, 19.7 nmol/L and 29.0 nmol/L. Two of the children were Caucasian, had normal body weight, but had low vitamin D intakes (< 3.0 ?g/day), and were seen during winter months. The other child had a plasma 25 (OH)D of 29.0 nmol/L and was a boy for whom the mother was South Indian, with a normal BMI and he was seen in July. This child did not take supplements, and had a vitamin D intake based on the FFQ, single 24 hr recall and three 24 hr recalls of 8.0 ?g/day, 2.7?g/day and 3.2 ?g/day, respectively. Differences in plasma 25 (OH)D concentrations across the four seasons for all children were analyzed and significant differences were found between winter and spring (P= 0.03), and between winter and fall (P= 0.013).  3.3.2 Plasma 25 (OH)D of children given and not given supplemental vitamin D during different seasons Notably, when children were divided into groups given or not given supplements, the significant differences in plasma 25 (OH)D across seasons discussed previously were no longer present among children given supplements (P= 0.100). Conversely, children not given supplements had significantly different plasma 25 (OH)D between winter (January ? March) and summer (July ? September) P= 0.014 (Table 3.13).        62  Table 3.73 Plasma 25 (OH)D as nmol/L for supplement users and non users divided into seasons  Supplement use is based on data collected by the FFQ ? Winter, spring, summer and fall include 40, 23, 20 and 21 children, respectively and children not given supplements include 13, 9, 9 and 15 children, respectively. * Indicate significant differences between seasons (P= 0.014).   Vitamin D intake and plasma 25 (OH)D The correlation coefficient between vitamin D intake estimated using the FFQ or three 24 hr recalls and plasma 25 (OH)D  was r= 0.39, P<0.001 and r= 0.3, P<0.001, respectively. No significant differences in plasma 25 (OH)D were found between children of mothers of different ethnicities (P= 0.187), and no relationship was found between 25 (OH)D, and bodyweight (P= 0.253) with a trend for BMI (P=0.062).         Supplemental vitamin D  No Yes   Mean ? SD Median (IQR)     Mean ? SD Median (IQR) Winter  49.8 ? 15.6 48.9* (8.1)  62.8 ? 15.8 60.9 (21.1) Spring  65.4 ? 13.5 67.6  (25.8)  71.8 ? 19.8 67.1 (31.7) Summer 73.1 ? 23.6 75.6* (23.5)  70.2 ? 12.6 67.8 (22.3) Fall 53.9 ? 14.2 53.7  (11.5)  63.7 ? 14.5 64.1 (26.5)  63  Chapter 4: Discussion When this study was designed, little information was available on whether vitamin D intakes below the vitamin D intake recommendations in young children would result in biochemical evidence of vitamin insufficiency or deficiency. Thus the main aims of this thesis were first to estimate vitamin D intake, the major sources contributing to vitamin D intake in young children, and the proportion of children meeting the EAR and RDA. Secondly, we determined the proportion of children who were vitamin D deficient, insufficient and sufficient, based on measure of plasma 25 (OH)D, and assessed the importance of season of the year and vitamin D intake as determinants of plasma 25 (OH)D.    4.1 Vitamin D intake This study found median vitamin D intake from foods below the EAR (10?g/day) across the three dietary collection methods in Canadian Children 5 ? 6 years of age. More specifically, the median vitamin D intakes from foods, excluding supplements, but including vitamin D fortified foods were 6.3 (IQR 4.6) ?g/day, 4.2 (IQR 4.6) ?g/day and 4.7 (IQR 3.5) ?g/day estimated using the FFQ, single 24 hr recall and three 24 hr recalls, respectively (Table 3.4). The intakes of vitamin D found in this study are lower than reported for data collected by the CCHS cycle 2.2 in 2004 which gave a median vitamin D intake from foods of 5.6?g/day (IQR 3.4), based on a single 24 hr recall 4. The difference in vitamin D intake between present study and the CCHS cycle 2.2 is about 1 ?g/day, and may reflect the group of children studied, changes in food intake patterns since the CCHS cycle 2.2, updates and changes to the nutrient databases used to analyze vitamin D intakes, and the dietary methodologies used. Updated DRIs for vitamin D were released in 20113 and these replaced the Average Intake (AI) of 5 ?g/day with an RDA of 15 ?g/day vitamin D, meaning that the DRI for vitamin D has tripled since the CCHS (2004). The results of the current study indicate that despite the new intake recommendations for  64  vitamin D, children?s vitamin D intake from foods has not increased appreciably over the last decade. Similarly, Hayek et al (2013) reported a daily median (IQR) vitamin D intake of 5.9 (4.2) ?g/d in Canadian children from Montr?al 6, which is also similar to the intakes found in the present study. As can be noted in Table 1.4 and Table 1.5, none of the studies of vitamin D intake among children in Canada or the U.S, including the present study, found mean or median dietary vitamin D intake above the EAR. This suggests that current food fortification practices do not enable children to meet the EAR for vitamin D. However, it is important to note that without fortification, vitamin D intake among young children would be exceedingly low. More specifically, the mean ? SD and median (IQR) vitamin D intakes from unfortified foods in the present study were only 2.0 ? 1.7 ?g/day and 1.4 (IQR 2.0) ?g/day and this is only about 20 % of the EAR (Table 3.9).    4.1.1 Vitamin D sources The main contributor to the daily dietary vitamin D was not unexpectedly milk, providing 62.6 % the daily dietary vitamin D intake while other dairy foods provided 3.0 % of the daily intake, based on data collected by the FFQ (Table 3.5).  The CCHS (2004) found that a slightly higher proportion of daily vitamin D intake was provided by milk products in children age 1 ? 8 years, namely 75.0 % compared to 62.6 % in the present study. Despite reporting a higher proportional intake of vitamin D from milk products, the mean ? SD intake from milk products in the CCHS (2004) was similar to the intake in the present study. We estimated a mean ? SD vitamin D intake from milk and dairy products of 4.5 ? 3.1 ?g/day, while the CCHS (2004) showed a mean intake of vitamin D from milk products of 4.7 ?g/day. Maguire et al (2013) reported a mean ? SD daily milk intake of 455 mL ? 307 mL day in 1 ? 5 year olds in Toronto, an amount which would provide 4.5 ?g/day ? 3.07 ?g/day vitamin D from milk 53. The latter  65  result is similar to our findings of 4.2 ? 3.1 ?g/day and median 3.7 (IQR 4.0) ?g/day vitamin D from milk based on data derived from the FFQ. Hayek et al (2013) reported 72.1 % of the daily vitamin D intake in children aged 2 ? 5 years in Montr?al was provided by milk. The children had a median intake of 2.4 servings of fluid milk/day, providing 6.0 ?g/day vitamin D from milk, also estimated using a FFQ 6. The latter estimate was higher than that found in our study, although the authors suggested that the high milk intake was likely due to implemented milk policies in Montr?al preschools in which children receive milk daily 6. Over all, there is agreement among studies that milk is the largest contributor to vitamin D intake in children, which was expected. Our study found fish to be the second largest source of daily vitamin D intake, providing 19.4 % of the daily intake. Similarly, Hayek et al (2013) found that fish provided 14.2 % of the daily vitamin D intake also estimated using a FFQ. In addition, we found that 63 % of the children did not eat margarine, and only 3 % of the daily vitamin D intake was provided by margarine. Thus, these results suggest that margarine is not a viable vehicle for increasing children?s vitamin D intake in our population. Over all, these studies indicate that obtaining vitamin D intakes to meet current recommendations from the diet can be difficult, and that the vitamin D intakes in children have not increased in recent years, regardless of the new higher intake recommendations.    4.1.2 Vitamin D intake from supplements The present study found that use of vitamin D containing supplements were frequent among young children with 57.5 % and 70.6 % of children given supplements at least two days/week, based on the three 24 hr recalls and the FFQ, respectively. More parents reported giving their child a supplement when asked using a FFQ than was recorded on three separate 24  66  hr recalls. All of the studies reported and discussed in the introduction to this thesis, collected information on the use of vitamin D supplements in children using a FFQ (Table 1.3). Thus, the following discussion is limited to results for supplement used based on the data gathered using the FFQ. The vitamin D intakes from supplements were highly skewed, mean ? SD 11.0 ? 9.3 ?g/day with a lower median of 10.0 (IQR 7.3) ?g/day (P<0.05), hence median intakes are used for discussion (Table 3.6). The median vitamin D intake provided by supplements among all children was 6.8 (IQR 10.0) ?g/day (Table 3.9). When children not taking supplements were excluded from the analysis, the median daily vitamin D intake from supplements for those children given supplements was 10.0 (IQR 7.3) ?g/day (Table 3.8). Hayek et al (2013) reported a somewhat lower median supplement dose of 7.1 (3.2 ? 10.0) ?g/day in children given supplements, also assessed using a FFQ. Maguire et al (2013) reported 57 % of 1898 children aged 1 - 5 years in Toronto were regularly taking vitamin D containing supplements, however, the study did not provide data on the quantity of vitamin D obtained from supplements 60. The CHMS 2007 ? 2009 estimated that 28.7 % of children aged 6 ? 11 years were given supplements 20, which is less than half of what we found in the present study (70.6 %). However, the CHMS (2007 ? 2009) was conducted before the release of new DRIs. Possibly, increased awareness of vitamin D in Canada has lead to an increased use of vitamin D containing supplements. Alternatively, differences in the group of children could be present in our study compared to the nationally representative CCHS (2004), or methodological differences could explain the differences in results. Similar to the CHMS (2007 - 2009), Hayek et al (2013) reported 27.7 % of 479 children aged 2 - 5 from Montreal were taking supplements 6. Stein et al (2006) showed that 61 % of Caucasian girls in Athens, in the US (34?N) aged 4 ? 8 years, were taking supplements56. Although there is variability in the proportion of children given vitamin D containing supplements across studies, it is still apparent that the practice of giving children  67  supplements is widespread. One exemption is the Canadian study by Hayek et al (2010), who found that only 3.7 % of Inuit preschoolers were reportedly given a vitamin D supplement, and 16.8% were given a MVI, whereas our study found 70.6 % of children were taking supplements. This may mean that there are subgroups in the population that are particularly susceptible to low vitamin D intakes, and these were not represented in the present study.   4.1.2.1 Total vitamin D intake The median (IQR) vitamin D intake from both foods and supplements reported by the CCHS (2004) was lower than in the present study, namely  8.2 (7.8) ?g/day 43 compared 12.4 (IQR 10.9) ?g/day in our study (Table 3.8). Our study found lower vitamin D intakes from the diet than the CCHS (2004), but higher intakes than the CCHS (2004) when considering vitamin D intakes from both diet and supplements. We found that supplements were the largest contributor to the overall vitamin D intake providing 52.8 % of the total daily vitamin D intake, but also with a large proportion of children given supplements (Table 3.9). A possible explanation for the lower vitamin D intake in the CCHS (2004) than the present study is that more children are now given supplements than a decade ago. Unfortunately, the CCHS did not include specific data on vitamin D supplement use among young children. Additionally, we found that fortified foods provided 33.1 % of the total vitamin D intake. Only 14.1 % of the total daily vitamin D intake was provided by natural food sources, corresponding to a mean ? SD and median (IQR) of 2.0 ? 1.7?g/day  and 1.4 (IQR 2.0) ?g/day, respectively. Notably, the combined intake of vitamin D from fortified foods and supplements provided 85.9 % of the total daily vitamin D intake based on the FFQ, corresponding to a mean ? SD and median of 12.2 ? 9.0 ?g/day and 10.8 (IQR 11.1) ?g/day (Table 3.9). In summary, we found that vitamin D intake from foods in our study was lower than that reported by the CCHS (2004), but the intake from  68  supplements was higher explained by a large proportion of children given supplements. Moreover, the results in this thesis indicate that children are reliant on vitamin D containing supplements to meet the current vitamin D intake recommendations, which will be discussed next    4.1.3 Vitamin D intake compared to the EAR and RDA  As discussed in section 3.2.4, vitamin D intakes from foods were low and the children?s estimated mean and median intakes did not meet the EAR (10 ?g/day) for any of the three dietary collection methods (Table 3.4). Analyzing vitamin D intakes from foods alone, in relation to the cut off for the EAR of 10 ?g/day, we estimated that 82.7 % and 90.7 % of the children did not meet the EAR based on data estimated using the FFQ and three 24 hr recalls, respectively (Figure 3.4A). When vitamin D intakes from supplements were included, the proportion of children not meeting the EAR decreased to 34.7 % and 51.4 % using data from the FFQ and three 24 hr recalls, respectively (Figure 3.4B). This again suggests that current vitamin D fortification practices do not enable children to reach the EAR, and are thus reliant on supplements to meet dietary recommendations. The importance of supplemental vitamin D intakes to enable children to reach the EAR separately was further emphasized when we compared vitamin D intakes to the EAR for children given supplements and children not given supplements. We found that 80.5 % and 89.5 % (estimated using the FFQ or three 24 hr recalls) of children not given supplements did not meet the EAR (Table 3.5 A). Conversely, of the children who were given supplements, only 15.7 % - 24.0 % did not meet the EAR (Table 3.5 A). Additionally, 93 % - 99 % of the children not given supplements did not meet the RDA for vitamin D compared to 43 % - 60 % for those given supplements (Table 3.5 B). This further adds to the evidence that the current food supply does not enable children to meet the current  69  intake recommendations for vitamin D, despite fortification of milk and margarine. Clearly, children not taking supplements seem to be at higher risk of not meeting the intake recommendations. These findings are concerning as food insecure individuals may potentially may have fewer resources to obtain supplements. The parents of the children included in the present study were predominately well-educated and many had an annual household income above $50,000. Whether they are representative of the general Canadian population or other groups is not known (Table 3.1). Notably, studies have suggested that individuals of higher socio-economic status (SES) are more likely to use supplements than individuals from lower SES61. Possibly, vitamin D intake from supplements in the general population may be lower than what is reported for children in this study.  When data from the CCHS was compared to the most recent DRI for vitamin D they showed that 59.8 % of children aged 4 - 8 years had vitamin D intakes below the EAR43, a result which was comparable to our data showing 60.1 % of children had intakes below the EAR, based on the same dietary and supplement collection method (i.e. single 24 hr recall and questionnaire on supplement use). Thus, it appears that even though we reported a higher total vitamin D intake compared to the CCHS (2004), the proportion of children not meeting the EAR was similar.    4.1.4 Vitamin D intakes assessed using FFQ and 24 hr recalls When planning nutritional research on dietary intakes it is important to consider the advantages and disadvantages of different dietary collection methods, and decide which method would be most effective in capturing what is intended to be investigated. It is difficult to estimate which method is more accurate, as the differences in results obtained with the FFQ compared to daily recalls may reflect overestimation on the FFQ or underestimation of average intakes on the  70  one or three day recalls. Throughout this thesis, it has been noted that energy intake as well as dietary and supplemental vitamin D intake were consistently higher when estimated using the FFQ compared to the 24 hr recalls.  This study also found that energy intakes were higher than Canada?s Food Guide?s average energy recommendations for 4 - 5 year old boys (1450 kcal/day) and girls (1350 kcal /day), particularly for the FFQ. The energy intake for girls estimated using the single 24 hr recall (1344 kcal) and the three 24 hr recalls (1388 kcal/day), compared closely to the Food Guide average energy intake recommendation for girls (1350 kcal/day), whereas the FFQ found much higher energy intakes of 1690 kcal/day.  For boys, the estimated energy intakes were 1833 kcal/day, 1651 and 1577kcal/day when estimated using the FFQ, single 24 hr recall and three 24 hr recalls, respectively (Table 3.3), again showing much greater energy intakes when using the FFQ. Since energy intakes using the single or three 24 hr recalls were closer to the Food Guide?s recommendations than the FFQ, it seems reasonable to suggest that the FFQ overestimated energy, and thus food, intakes.  Data derived from the FFQ showed a 25.4 % higher vitamin D intake than when estimated as the average of three 24 hr recalls, (P <0.001). However, after adjusting for energy intake the difference between the methods decreased to 13.5 %, although, vitamin D intakes estimated using the FFQ were still significantly higher than when estimated using the three 24 hr recalls (P= 0.006) (Table 3.4). The difference in vitamin D intake between boys and girls was not significantly different after adjustment for energy intake across methods. The higher vitamin D intake estimated from the FFQ compared to dietary recalls even after adjustment for energy intake could be explained by the methodology, for example rounding of portion sizes or frequency on the FFQ, but not the recalls. A 24 hr recall assesses vitamin D intake on a particular day, but foods not eaten on a regular basis are not always captured by a recall. The  71  FFQ, however, is designed to capture the foods not eaten daily, such as fish which might provide a relatively large amount of vitamin D in a single serving. The inclusion of foods that provide significant amounts of vitamin D but are not eaten daily would be expected to result in higher estimates of vitamin D intake using a FFQ than a single 24 hr recall or three 24 hr recalls, even after energy adjustment. As an example, Mulder (2013) explored the proportion of children (n= 188), based on same data set as this thesis, who were classified as fish eaters when estimated by the FFQ, single 24 hr recall or three 24 hr recalls. Notably, 82.4 % of children were classified as consumers of fish using the FFQ, but only 37.9 % and 16.7 % of the children were classified as fish eaters when using three or one 24 hour recall, respectively62. Based on this, data collected with the FFQ was used to estimate the proportion vitamin D intake provided by different foods for the children in this study with the aim of ensuring that foods not eaten on a regular basis, such as fish and egg, would be included (Table 3.5).  4.2 Plasma 25 (OH)D The mean ? SD and median (IQR) plasma 25 (OH)D for all children in this study were above 50 nmol/L, namely 64.4 ? 17.3 nmol/L and 62.2 (IQR 24.0) nmol/L, respectively (Table 3.10). A recent study published by Maguire et al (2013) reported a mean 25 (OH)D of 88 nmol/L in children aged 2 ? 5 years from Toronto 60, which is substantially higher than our findings. The latter study found that only 6 % of the children were classified as vitamin D insufficient (plasma 25 (OH)D < 50 nmol/L). Similarly, Hayek et al (2013), reported a median plasma 25 (OH)D of 74.4 nmol/L in children aged 2 ? 5 years of age in Montreal, and only 12.0 % of the children had a plasma 25 (OH)D below 50 nmol/L6. Similar to Hayek et al (2013), the present study found that 81.2 % of the children were vitamin D sufficient (plasma 25 (OH)D > 50 nmol/L), 16.9 % were insufficient , and only 1.9 % had a 25 (OH)D in the deficient range (plasma 25 (OH)D < 30  72  nmol/L). This is also comparable to data reported by the CHMS (2007 ? 2009) in which 14.1 % of children aged 6 ? 11 years were classified as vitamin D insufficient20 . The CHMS (2009 ? 2011) reported a mean 25 (OH)D of 73.9 nmol/L for 3 - 5 year old children, and 67.3 nmol/L for 6 ? 11 year old children, which corresponded to 11.0 % and 24 % of the 3 ? 5 year old and 6 - 11 year old having plasma 25 (OH)D below 50 nmol/L, respectively45. Comparison of our study and those of Maguire et al (2013) and Hayek et al (2013) suggest there has not been a substantial positive change in vitamin D status in children between 6 and 11 years since the most recent DRIs for vitamin D and calcium were published. As data for 3 ? 5 year old children were not included in the CHMS 2007 ? 2009, comparison to that national survey cannot be made. However, it appears that younger children have higher 25 (OH)D than children over the age of 6 years, as few children 3 ? 5 years of age were vitamin D insufficient (11 %) in the CHMS (2009 ? 2011).     4.2.1 Season   We found significant differences in plasma 25 (OH)D across seasons (Table 3.12), which was not explained by differences in vitamin D intake (Table3.11). However, when the children were split into those given or not given supplements, differences in vitamin D status were evident across seasons in the non-supplement users, but not in the supplement users (Table 3.13). These results suggest that supplement use overcomes any effect of season on vitamin D status. Similar findings were noted by Whiting et al (2011) using data from the CHMS (2007 ? 2009). Interestingly, Whiting et al (2011) showed much higher mean plasma 25 (OH)D in both supplement users and non users in winter than the values in our study. They found that in children aged 6 ? 11 years, the mean plasma 25 (OH)D was 83.1 nmol/L in supplement users and 67.9 nmol/L in non-users during winter months (defined as October - March). We found a  73  substantially lower mean ? SD 25 (OH)D concentration of 62.8 nmol/L and 63.7 nmol/L in supplement users during winter and fall months (Jan ?Mar and Oct ? Dec) and mean plasma 25 (OH)D in non ? supplement users of 49.8 nmol/L and 53.9 nmol/L during winter and fall months, respectively.  The CHMS did not include quantifiable vitamin D intake data, which complicates interpretation of the differences in plasma 25 (OH)D between studies, although several suggestions can be made.  Overall, our study conducted in Vancouver found lower 25 (OH)D than found in the CHMS (2007 ? 2009). Whether this is reflective of climate, higher use of sun screen or clothing to protect from sun exposure, or methodology to measure 25 (OH)D is not known.   4.2.2 Relationship of vitamin D intake to vitamin D status  As described previously, the IOM (2011) concluded that vitamin D intakes of 10?g/day (EAR) and 15 ?g/day (RDA) would achieve 25 (OH)D plasma concentrations of approximately 40 nmol/L and 50 nmol/L, respectively. However, Hayek et al (2013) suggested that these vitamin D intake recommendations could be set too high for children, as they found that 95 % of the children aged 2 ? 5 years had intakes below the EAR, but only 4.5 % had a plasma 25 (OH)D below 40 nmol/L. However, they noted that sunlight could have been a confounding factor, which they did not control for6. Additionally, Cashman et al (2011) conducted a study with adolescent girls, estimating the vitamin D intakes level required to reach a plasma 25 (OH)D of 40 nmol/L or 50 nmol/L. They estimated that a plasma 25 (OH)D > 40 nmol/L could be reached with vitamin D intakes of 6.3?g/d. However, they suggested that an intake of 18.6 ?g/d was needed to reach a plasma concentration above 50 nmol/L5. In this thesis, we estimated that 35.8 % of the children who provided complete dietary data and a blood sample, had vitamin D intakes below the EAR and 57.4 % had intakes below the RDA, estimated using the FFQ. Similar to  74  Hayek et al (2013), we found that only 4.7 % the children had a 25 (OH)D below 40 nmol/L, and only 19.6 % had a 25 (OH)D below 50 nmol/L. As was noted by Hayek et al (2013), sun exposure could be a confounding factor6. Further, the IOM (2011) developed their intake recommendations assuming minimal sun exposure3. Some insight into this may be gained by considering vitamin D intakes and status during the non vitamin D synthesizing months (Jan ? Mar and Oct ? Dec). During the non-synthesizing months (defined as January - April and October - December), 87 children provided both dietary data and a blood sample in the present study. Of these children, 54 met the EAR and 33 did not meet the EAR for vitamin D. Of the children who met the EAR, only 3.6 % had a 25 (OH)D concentration below 40 nmol/L. Unexpectedly, we showed that only 12.5 % of the children not meeting the EAR for vitamin D had a 25 (OH)D below 40 nmol/L in the non synthesizing months (Figure 4.1 A). Of the children who had vitamin D intakes below the RDA, 40.0 % had a plasma 25(OH)D below 50 nmol/L (Figure 4.1 B). It was surprising that a large proportion of children who had vitamin D intakes below the EAR had a plasma 25 (OH)D above 40 nmol/L, even during winter months. This suggests that the EAR for vitamin D intake in children could is high, as suggested by Hayek et al (2013). The intake recommendations set by the IOM (2011) were based mainly on studies conducted in adults, but also adolescents and older children, although none of the studies included children below the age of 6 years 3. The dose response relationship for vitamin D in children is currently unknown, and thus it is not clear whether children can consume less vitamin D than adults but still reach same plasma 25 (OH)D. Further, information on the efficiency of absorption and cutaneous synthesis in children are lacking, and it is possible that both are more efficient in young children than adults.    75  Figure 4.1 Children with vitamin D intakes below or above EAR (10 ?g/day) or RDA (15 ?g/day) with a plasma 25 (OH)D above or below 40 nmol/L or 50 nmol/L in winter months     Results shown are dietary and supplement data derived from the FFQ. Non- synthesizing months are defined as January ? March and October ? December. A). Proportion of children below or above 40 nmol/L with intakes < EAR (n= 32) or > EAR (n= 55), EAR = 10 ?g/day vitamin D. B). Proportion of children below or above the RDA with intakes < RDA (n= 50) or < RDA (n= 37), RDA = 15?g/day vitamin D.  76  There is little information available regarding 25 (OH)D concentrations and health outcomes in children, and no final consensus on vitamin D status cut off concentrations for deficiency, insufficiency and sufficiency, have been reached. Some studies have suggested higher cut-off values for 25 (OH)D 40?42,63, namely 75 nmol/L. If the cut-off values were changed to 75 nmol/L, 27.3 % of the children in our study would have a plasma 25 (OH)D above 75 nmol/L, and thus 72.7 % would be considered insufficient. Most of the studies used to suggest a cut-off of 75 nmol/L were based on studies in adults, not children. Studies are needed to assist in defining 25 (OH)D plasma levels for health outcomes especially in children, as well as the dose-response relationship between vitamin D intake and 25 (OH)D in children, efficiency of vitamin D absorption and cutaneous synthesis. Further, studies exploring whether vitamin D intake recommendations for children should be based on body weight might also be useful.   4.3 Strengths and limitations This study was conducted in Vancouver, and may not be representative of the entire Canadian population, subgroups not represented in this study, or children of other ages, or with health problems. Further, our study used a volunteer sample, who might not be representative as individuals who enroll in nutrition research may be more health or diet conscious or have different dietary patterns than individuals who do not volunteer for research. In addition, individuals with higher incomes or education might eat differently from those with lower incomes or education, as they may have more resources or knowledge. It could also be speculated that individuals with lower income and knowledge do not volunteer to participate in a nutrition study that gathers this type of information.  The FFQ utilized in this study was not validated for vitamin D, but was designed to gather dietary information on fat. However, as vitamin D is a fat soluble vitamin, the foods  77  included and emphasized in the questionnaire are all those that also provide vitamin D. Further, the FFQ records answers to what is being asked, and if some foods are not included in the FFQ they may be omitted, although spaces are available to record additional foods (Appendix C). However, the FFQ does capture vitamin D containing foods not eaten on a regular (daily) basis and is therefore a good method to use when investigating nutrients not present in staple foods eaten daily. This study also included 24 hr recalls, which do not need to be validated like the FFQ, although they are also subject to reporting errors and bias.   One important limitation of this study is the accuracy of supplement intake data. The parents often did not report the dose of the vitamin given (i.e. sometimes one pill/gummy is recommended by the manufacturer and sometimes two) and an assumption was made that the dose was the same as that recommended for the brand. Further, the answers for frequency with which supplements were given were often not clear, perhaps because parents sometimes forget or other activities were occurring. A prospective calendar might help overcome inaccuracies in reporting the frequency, amount and type of supplements given to children. In sum, the supplement data may lack precision, and should be used with caution, although it may provide information on trends on supplement use or awareness.  We did not collect information on sun exposure, we merely recorded the month the child attended the research study, and blood was collected. We also did not collect information on whether the child had been on vacation somewhere sunny during winter months, or hours of outdoor play. We used the ethnicity of the mother as a proxy for child ethnicity, and also did not collect specific information on the child?s skin color.  It is also important to acknowledge when comparing data on the estimated vitamin D across different studies that the food supply is constantly changing, and that data bases for estimating micro and macro nutrients are also updated. In this regard, we did not collect  78  comprehensive data on yogurt brands, which would have been helpful as some yogurts are now made with vitamin D fortified milk. At the onset of this study in July 2010, few yogurts were manufactured using fortified milk, so information on specific brands was not a major priority, and only information on fat content was collected. However, during the course of the study some but not all brands of yogurt changed to use vitamin D fortified milk, but these appeared on the market and in different stores at different times. Also important, these changes in the vitamin D content of yogurts had not been made in the Canadian Nutrient File at the time of completion of this thesis.   Differences in analytical methods for quantifying 25 (OH)D concentrations should be considered when comparing data on vitamin D status from different studies. This study used LC-MS/MS whereas many of the studies reviewed in this thesis used an immunoassay. The immunoassay is less specific than the LC-MS/MS as it does not differentiate between the different vitamin D metabolites. The LC-MS/MS uses the mass of the molecule to quantify the concentrations, thus only 25 (OH)D3 was measured, making the method more specific.    4.4 Future directions This study has suggested that the EAR may be set too high for vitamin D intakes for young children, although little is known about vitamin D status and its relation to health outcomes in this age group. Studies are needed to assist in defining 25 (OH)D plasma levels for health outcomes, beyond clinically identifiable rickets. Dose-response studies to define the relationship between vitamin D intake and 25 (OH)D in children, efficiency of vitamin D absorption and cutaneous synthesis should be investigated. This would help understand whether children have the same vitamin D requirements as adults.    79  Chapter 5:  Conclusion This study has shown that the vitamin D intake in 5 - 6 year old children living in Vancouver BC are below the EAR and RDA. No evidence of an increase in vitamin D intakes from food sources were apparent when compared to published data on vitamin D intakes of Canadian children, despite release of the new DRIs. The current fortification of milk and margarine do not enable children to meet their vitamin D intake recommendations from foods. The results suggest that vitamin D intake from supplements may have increased in children in recent years and that children may need vitamin D containing supplements in order to reach the EAR and RDA for vitamin D.   However, even though vitamin D intakes from both foods and supplements appears insufficient, this was not reflected in plasma 25 (OH)D in the deficient or insufficient range. Only 12.5 % of children not meeting the EAR had plasma 25 (OH)D below 40 nmol/L, even during the non-vitamin D synthesizing months, indicating that the EAR may be set too high for children. 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Modifiable determinants of serum 25-hydroxyvitamin D status in early childhood: opportunities for prevention. JAMA Pediatr 2013; 167: 230-235. 61. Vatanparast H, Adolphe JL, Whiting SJ. Socio-economic status and vitamin/ mineral supplement use in Canada. Health Rep 2010; 21: 19-25. 62. Mulder KA. Unpublished data: Fish consuption besed on three different dietary methods; 2013.  86  63. Bischoff-Ferrari HA, Giovannucci E, Willett WC, Dietrich T, Dawson-Hughes B. Estimation of optimal serum concentrations of 25-hydroxyvitamin D for multiple health outcomes. Am J Clin Nutr 2006; 84: 18-28.                        87  Appendices  Appendix A Informed consent form  INFORMED CONSENT AND SUBJECT INFORMATION    n-3 Fatty Acids and Early Child Nutrition: 5 Year Old Follow-Up  1. Introduction and Background Information  The links between food choices and healthy development of children is complex and not well understood. Several studies suggest that some fats found in certain foods, called n-3 fats, may influence child development and health, including how well children do in school, and the risks of developing overweight, diabetes and allergies. The links appear to be related to the n-3 fats the mother eats during pregnancy as well as the n-3 fats that children eat themselves. But not all studies agree.  It is known that many things influence what young children eat.  Sometimes children have strong likes and dislikes, are more comfortable eating a few familiar foods or refuse to try new foods. These things may influence how much n-3 fats children eat, as well as other nutrients important to child health, such as iron, folate, B12  and vitamin D.  There is also new information that there are differences in the way people handle fats and that these differences may be explained by small differences in the genetic make-up also known as the DNA of individuals. We are inviting the mothers of children who participated in our previous study on fats called n-3 fatty acids during pregnancy to participate in this study. We are trying to understand the importance of n-3 fats to children?s health and development, how children differ with respect to how much of the n-3 fats they need and some of the reasons why certain foods are and are not eaten by 5 year old children.  Together with our previous study in which mothers participated with their infants to 18 months of age, this study will provide important information on how the fats in our diet contribute to healthy child growth and development and whether changes are needed to improve the types of foods available for children.  2.   Your Participation is Voluntary  Your participation is entirely voluntary, so it is up to you to decide whether or not to take part in  88  the study. Before you decide, it is important for you to understand what the research involves. This consent form will describe 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 participate, you will be asked to sign this consent form. If you decide to take part in this study, you are still free to withdraw at any time and without giving any reasons for your decision(s). If you do not wish to participate, you do not have to provide any reason for your decision(s) 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 and friends before deciding.   3.  Who is Conducting the Study?  The study is being conducted by the Nutrition Research Program of the Child and Family Research Institute, University of British Columbia. Funding has been received from the Canada Institute of Health Research to complete this study and you are entitled to request details about the research funding from the Principal Investigator.   4.  What is The Purpose of the Study?  We will gather information on what you and your 5 year old child are eating and some measures of growth and development. A blood sample will be used to measure n-3 fats and other nutrients related to health. Because people with the same fat intakes can have different blood levels of the n-3 fats, we will also look at the DNA, to measure the differences in the genes that make the proteins and enzymes that may affect the blood levels of the n-3 fats.   5.  Who Can Participate?  To be eligible you must:  ? have previously participated in the research study  we conducted called: n-3 Fatty Acid Requirements for Human Development ? be able to speak and read  English fluently   ? be the mother or primary caregiver  of a  child  5 ? 6 years of age  89  6.  Who Should Not Participate?   You may not participate in this study if you have not previously participated in the research study we conducted called: n-3 Fatty Acid Requirements for Human Development.   7. What Does the Study Involve?  This study will be conducted at the Oak Street Campus of UBC at the Child and Family Research Institute (CFRI) or other suitable and convenient locations. If you agree to participate in this study, then you will be asked to complete the study procedures described below. This will take about 2-3? total hours of time to complete.  There are parts of the study that do not require your child be present. This means that if you prefer and it is more convenient, your participation can be separated into two visits, one of which will involve your child. The research assistant will be able to give more detail and answer any questions you may have.   Study Procedures:  If you agree to participate in this study then you will:  ? Update the general information you provided in our previous study by completing questionnaires asking about your age, weight, ethnic background, education, number of adults and children in your household, household annual income, and provide some information on factors that influences food choices in your household. You are under no obligation to answer any questions or provide information that makes you feel uncomfortable.   ? Repeat the puzzle like activity that involves solving what comes next in a series of different shapes.  ? Complete a brief questionnaire on your child?s general health that includes some questions on allergies.  ? Provide information to describe the foods and beverages you and your preschool age child eat.  This will involve telling us everything that your child ate and drank for one entire day (called a 24-hour recall) and answering some questions about how often you  90  and your child eat and drink some foods and beverages (called a food frequency questionnaire). This will take about 30 minutes of your time. The foods we eat and drink are not the same every day. To get a better idea of your child?s diet, we will telephone you two times at a time that you tell us is convenient and each time we will ask you to tell us everything your child ate and drank the day before. This should take about 10-15 minutes. Your child does not need to be present when you tell us about your child?s diet and general health. When this is complete, a second appointment will be made for about 1-2 weeks later for you and your child.  ? Allow your child to complete some play like activities that will assess attention, language and motor skills which will take about 90 minutes to complete. The best time for this is in the morning as young children are often tired later in the day.  ? Allow us to measure your child?s skin-fold thickness, weight and height.   ? Allow some blood (6 mL = 1 teaspoon) to be drawn, and heart rate and blood pressure to be measured on your child at the end of the appointment. We will use this blood sample to study the levels of n-3 fats, some nutrients related to nutritional health and the differences in genes which make the proteins and enzymes affecting the blood levels of the n-3 fats.  ? Allow some blood (6 mL = 1 teaspoon) to be drawn, and heart rate and blood pressure to be measured on yourself at the end of the appointment. We will use this blood sample to study the levels of n-3 fats, some nutrients related to nutritional health and the differences in genes which make the proteins and enzymes that may affect the blood levels of the n-3 fats. ? Allow us to hold a meter called a Chromameter close to your skin and your child?s skin at the upper underarm (which is not generally exposed to sunlight) and the back of the hand (which is generally exposed to sunlight). The meter will measure the amount of light reflected from the skin. The readings will take about 10 seconds to complete.  91   8.  What are the possible risks or harms of participating in this study?  We are not aware of any risks or harms that will result from participating in this study. A certified technologist or registered nurse will draw a small amount of blood. The needle used to take blood might feel uncomfortable, result in feeling faint, lightheaded or dizzy and might cause some minor bruising, or rarely an infection at the site of the blood draw. The blood samples provided by you will not be used for any purposes other than the research in this study.   9.  What are the benefits of participating in this study?  There may not be direct benefits to you from taking part in this study. We hope that the information learned from this study can be used in the future to improve dietary recommendations for all infants and preschool children. In the event that any of the nutrients that are measured are outside the normal ranges, then we can provide a report for you to follow-up with your doctor. If needed we will reimburse any costs associated with travel to participate in this study. In appreciation of the time that it takes to complete this study you may choose to receive up-to-date nutrition education materials and an age appropriate gift for your child.  10.  What will happen if I decide not to participate or wish to withdraw from this study?  Your participation in this research is entirely voluntary. You may withdraw from this study at any time and without providing any reasons for your decision. If you decide to enter the study and then withdraw, there will be no penalty or loss of benefits, if any, to which you are otherwise entitled. The study investigators may decide to discontinue the study at any time, or withdraw you from the study at any time, if they feel that it is in your best interests. If you choose to enter the study and then decide to withdraw at a later time, all data collected about you during the enrolment part of the study will be retained for analysis since by law, this data cannot be destroyed for a period of five years, after which the study information may be shredded.      92  11.  How will my participation be kept confidential?  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 UBC 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. In this study your samples and questionnaires will be identified by a study code and any identifying information will be kept in a locked and secure area. Study code numbers are not linked to any personal, healthcare or other identifying information and results of the blood tests and clinical records will not become part of your health record. The blood samples in this study will not be used for any purposes other than the research in this study and once the study is completed the blood samples will be destroyed. No information that identifies you will be allowed to leave the study center or be used in any reports or publications about the study. Signing this consent form in no way limits your legal rights against the sponsor, investigators, or anyone else.  12.  What if I have concerns about my rights as a study subject, have questions or want more information? 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 the information. Further details about this legislation are available on request. 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 UBC Office of Research Services at phone number: X.  93  This study will be explained to you and you will be given the opportunity to ask questions. If you have questions or want more information about the study procedures before or during participation, you may contact us at any time. 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. ? You and your child agree to take part in this study.  13.  Signature/Consent to Participate:    My signature on this consent form means that I:  ? have had this study explained to me, read this form and understand the information concerning this study. ? have had sufficient time to consider the information provided, get advice and ask questions if  necessary and  I have received satisfactory responses to my questions. ? understand that all of the information collected will be kept confidential and that the results will only be used for scientific objectives. ? understand that my 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 giving any reason(s) and my decision to withdraw will not change in any way the quality of care that I receive. ? understand that signing this consent form in no way limits my legal rights against the sponsor, investigators or anyone else. ? understand that there is no guarantee that this study will provide any benefits to me. ? understand that if I have any further questions or desire further information I should contact Dr X or one of her associates at phone number X. ? understand that if I have any concerns about my rights or the rights of my child as a research subject or my experiences while participating in this study, I may contact the  94  Research Subject Information Line located in the UBC Office of Research Services at phone number X. ?  have been told that I will receive a dated and signed copy of this form for my own record. ? (Optional) I understand that there may be follow-up studies on n-3 Fatty Acids & Early Child Nutrition and I may be contacted to determine my interest in participating  I, ___________________________________    voluntarily give consent for my and my child?s      (Subject-Parent-Guardian.  Please print your name)    ___________________________________ participation in the research study entitled:             (Please print child?s name)  n-3 Fatty Acids and Early Child Nutrition:  5 Yr Follow-Up.           ________________________________________________        Signature of Subject-Parent-Guardian or person legally authorized to give consent           ________________________________________________         ____________________ Relationship to Child (Mother, father, legal guardian etc)               Date    _______________________     _______________________       ___________________ Witness Signature         Printed Name                 Date _______________________                                                       ___________________ Investigator Signature Printed Name of principal investigator       Date           95  Appendix B Socio-demographic questionnaire  Socio-Demographic and Health Questionnaire 5 Year Old Follow-Up Date:          ID #:      Please answer questions as accurately as possible. If you are unsure of an answer, please leave it blank. Do not guess. Your answers will be kept strictly confidential. Remember that your name is not attached to any of these questions.   1. What is your age?: ? Less than 20 years ? 20-29 ? 30-39 ? 40-49 ? Over 50  2. Please check all that you completed: ? High school ? College/vocational diploma ? University undergraduate degree ? University graduate degree       3. What is your current height:_________ weight : ________  4. How long have you lived in Canada?:  ?  always               ? ______ years  5. My cultural/ethnic background is best described as:   ? White/Caucasian      ? First Nations      ? Black      ? Chinese       ? East Indian      ? Asian Other ______________  ? Other: ___________________        96  6. In addition to caring for your child, do you have another occupation?  Please describe:   7. What is the total annual income in your household? ? Under $30,000  ? $30,001-50,000 ? Over $50,000  8. How many persons live in your household?         Adults:   ______      Children over 5 years: ______ Children under 5 years: _____                              97  Appendix C Food frequency questionnaire    98       99    100    101     102    103     104    105     106      107            108        109     110   Does this child take any vitamin, mineral and/or other dietary supplement(s)? Please tell us as much as you can about the supplement, brand, and how often.  ? No         ? Yes:  ? Supplement name       ? How often do they take them?     ? When did they start taking them?     ?  Why did they start taking them? _______________________________________                         

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