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Vitamin D status of pregnant women in Vancouver Li, Wangyang 2010

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VITAMIN D STATUS OF PREGNANT WOMEN IN VANCOUVER by Wangyang Li B.Sc. (Honours), Acadia University, 2008 A THESIS SUBMITrED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE m The Faculty of Graduate Studies (Human Nutrition) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2010 © Wangyang Li, 2010 ABSTRACT Obtaining adequate vitamin D during pregnancy is important for health of both mother and child. Low 25-hydroxyvitamin D (250HD) concentrations, the best indicator of vitamin D status, have been reported in pregnant women in several countries; yet, there are few studies of pregnant women in Canada. We determined vitamin D status, based on 250HD concentrations, of pregnant women living in Vancouver and explored the determinants of 250HD such as ethnicity, season and vitamin D intake. A convenience sample of 336 pregnant women was recruited from Greater Vancouver (49°N). Participants completed a demographic and lifestyle questionnaire as well as a food frequency questionnaire to estimate vitamin D intake. A blood sample was collected and analyzed for plasma 250HD and parathyroid hormone (PTH). Overall the mean (95% CI) 250HD was 66.7 (64.2, 69.1) nmol!L. Only 1% of women had a 250HD concentration indicative ofdeficiency (<25 nmoifL). Howevei; 24% and 65% of women were vitamin D insufficient based on cutoffs of 50 and 75 nmoiIL, respectively. Over 90% of women took vitamin D containing supplements and the median intake of vitamin D from food and supplements was 16.0 jig/d. In adjusted analysis, women of European ethnicity had higher mean 250HD concentrations than women of Other ethnicity [69.1 (62.8, 75.3) cf. 59.0 (52.2, 65.8) nmoIfL; P = 0.004]. However, there were no differences between Europeans, Chinese, and South Asians. Mean 250HD was lower in winter [55.1 (47.7, 62.5) nmol!L] than spring [64.7 (57.7, 71.7) nmol!L] and summer [67.4 (59.9, 74.9) nmol/L] but not fall [63.2 (55.6, 70.8) nmoIJL]. Only 5% of women had elevated PTH concentrations (>6.4 pmol!L) and plasma PTH was only weakly inversely related to 250111) (R2 = 0.034; P = 0.001) with no apparent inflection point. Despite high supplement use, vitamin D insufficiency appears to be common in this group of pregnant women. Ethnicity and season were determinants of 250HD but the 11 magnitude of their effect was less pronounced than in other studies. Strategies to improve the vitamin D status ofpregnant women may be required, such as increasing the amount of vitamin D in maternal supplements. 111 TABLE OF CONTENTS ABSTRACT.ii TABLE OF CONTENTS iv LIST OF TABLES vi LIST OF FIGURES vii ACKNOWLEDGEMENTS viii CHAPTER 1: INTRODUCTION 1 CHAPTER 2: LITERATURE REVIEW 5 2.1 Vitamin D Metabolism 5 2.2 Assessment of Vitamin D Status 7 2.3 Defining Vitamin D Deficiency and Insufficiency 8 2.3.1 Parathyroid hormone suppression 9 2.3.2 Calcium absorption 10 2.3.3 Bone density and facture risk 11 2.4 Factors Associated with 25-Hydroxyvitamin D Concentration 12 2.4.1 Environmental conditions: latitude, season and climate 13 2.4.2 Skin color and ethnicity 14 2.4.3 Tanning, sun avoidance, sunscreen and clothing 16 2.4.4 Obesity 18 2.4.5 Diet and fortification 19 2.5 Vitamin D Status of Pregnant and Lactating Women 20 2.5.1 Canada 20 2.5.2 United States ofAmerica 22 2.5.3 Other countries 24 2.6 Current Recommendations for Intake of Vitamin D for Pregnant and Lactating Women 25 2.6.1 Nutrient reference values 25 2.6.2 Tolerable upper level of intake and toxicity 26 2.6.3 Vitamin D intake from food sources and supplements 27 2.7 Consequences of Inadequate Vitamin D during Pregnancy and Lactation.. 29 2.7.1 Vitamin D metabolism during pregnancy and lactation 29 2.7.2 Musculo-skeletal health of mothers 31 2.7.3 Non-skeletal health of mothers 32 2.7.3.1 Preeclampsia 32 2.7.3.2 Gestational diabetes 33 2.7.4 Musculo-skeletal health of infants 34 2.7.4.1 Rickets 35 2.7.5 Non-skeletal health of infants 37 2.7.5.1 Type idiabetes 37 2.7.5.2 Multiple sclerosis 38 2.7.5.3 Infant birth weight 39 2.7.5.4 Mental disorders 40 2.8 Vitamin D Supplementation during Pregnancy and Lactation 41 2.8.1 Impact of vitamin D supplementation during pregnancy on maternal 25-hydroxyvitamin D concentrations and neonatal outcomes 41 2.8.2 Vitamin D supplementation during lactation 43 CHAPTER 3: METHDOLOGY 62 3.1 Overview of Design 62 3.2 Sample Size 62 iv 3.3 Recruitment and Participant Selection.62 3.4 Procedures 64 3.4.1 Assessment of dietary intake 64 3.4.2 Skin color measurement 65 3.4.3 Blood collection and laboratory methods 66 3.5 Data Analyses 67 3.6 Miscellaneous 69 CHAPTER 4: RESULTS 70 4.1 Recruitment and Participant Characteristics 70 4.2 Sun Exposure 71 4.3 Vitamin D Intake 71 4.4 Biochemical Outcome Measures 72 4.5 Relationship between 25-Hydroxyvmtain D and Parathyroid Hormone Concentrations 75 4.6 25-Hydroxyvmtain D, Parathyroid Hormone and Calcium Intake 75 4.7 Skin Color 75 CHAPTER 5: DISCUSSION AND CONCLUSIONS 97 5.1 VitaminDintake 97 5.2 Vitamin D Status and Prevalence of Inadequacy 101 5.3 Factors Associated with 25-Hydroxyvitamin D Concentrations 103 5.4 Effect of Skin Color 108 5.5 Relationship between 25-Hydroxyvitamin D and Parathyroid Hormone Concentrations 109 5.6 Effect of Calcium Intake on 25-Hydroxyvitamin D and Parathyroid Hormone 113 5.7 Limitations 114 5.8 Directions for Future Research 115 5.9 Conclusions 117 REFERENCES 119 APPENDICES 141 Appendix A: University of British Columbia Ethics Approval 141 Appendix B: Children’s & Women’s Health Center of British Columbia Ethics Approval 142 Appendix C: Consent Form 143 Appendix D: Recruitment Brochure 148 Appendix E: Recruitment Poster 150 Appendix F: Demographic and Lifestyle Questionnaire 151 Appendix G: Food Frequency Questionnaire 156 Appendix H: Participant Thank You Letter 159 V LIST OF TABLES Table 2.1 Food sources of vitaminD.46 Table 2.2 Vitamin D status of pregnant and lactating women 47 Table 2.3 Variation in recommended vitamin D intake for (non-high-risk) pregnant and lactating women 52 Table 2.4 Summaiy of vitamin D supplementation studies during pregnancy 53 Table 2.5 Summary of vitamin D supplementation studies during lactation 55 Table 4.1 Participant recruitment 78 Table 4.2 Characteristics of participants 79 Table 4.3 Time spent outdoors during work days and leisure days 80 Table 4.4 Outdoor clothing practice over the past two months 80 Table 4.5 Sunscreen use, sun bathing, and sun bed use by season 81 Table 4.6 Vitamin D supplement use by age, etlmicity, season, and education 82 Table 4.7 Vitamin D intake (.tg/d) from diet and supplement by age, ethnicity, season, and body mass index 83 Table 4.8 Percentiles ofplasma 25-hydroxyvitamin D by age, week of gestation and ethnicity 84 Table 4.9 Plasma 25-hydroxyvitamin D concentrations and prevalence of insufficiency 85 Table 4.10 Adjusted mean plasma 25-hydroxyvitamin D concentrations and prevalence of insufficiency 86 Table 4.11 Estimated difference in 25-hydroxyvitamin D according to selected variables 87 Table 4.12 Mean plasma parathyroid hormone concentrations 88 Table 4.13 Exposed (outer forearm) and unexposed (upper inner ann) skin color classification by ethnicity 89 Table 4.14 Distribution of variables of unexposed (upper inner arm) and exposed (outer forearm) skin color by ethnicity 90 Table 4.15 Adjusted plasma mean 25-hydroxyvitamin D concentrations by skin color classification 91 vi LIST OF FIGURES Figure 2.1 The chemical structure of vitamin D2 (ergocalciferol) 58 Figure 2.2 The chemical structure of vitamin D3 58 Figure 2.3 Schematic diagram of cutaneous production of vitamin D and its metabolism and regulation for calcium homeostasis and cellular growth. .59 Figure 2.4 The chemical structure of calcitriol (1,25 dihydroxyvitamm) 60 Figure 2.5 The chemical structure of calcidiol (25-hydroxyvitamin D) 60 Figure 2.6 Serum 25-hydroxyvitamin D concentrations by month, of New Zealand children and adolescents (n = 1583) who participated in the 2002 National Children’s Nutrition Survey 61 Figure 4.1 Histogram of plasma 25-hydroxyvitamin D concentration 92 Figure 4.2 Mean (95% CI) plasma 25-hydroxyvitamin D concentrations by season, adjusted for age, week of gestation, ethnicity, pre-pregnancy body mass index, smoking, and total vitamin D intake 93 Figure 4.3 Mean (95% CI) plasma 25-hydroxyvitamin D concentrations by month, adjusted for ethnicity 93 Figure 4.4 Association of plasma parathyroid hormone with 25-hydroxyvitamin D concentration 94 Figure 4.5 Mean (95% CI) plasma parathyroid hormone concentration by tertile of plasma 25-hydroxyvitamin D concentrations and tertile of calcium intake (mg/d) 95 Figure 4.6 Distribution of study participants by exposed skin color 96 Figure 4.7 Distribution of study participants by unexposed skin color 96 vii ACKNOWLEDGEMENTS I would like to acknowledge my thesis supervisor Dr. Tim Green for his guidance, honesty, patience, encouragement, enthusiasm and fmancial support throughout this journey. I would also like to thank my thesis committee members Drs. Susan Barr and Sheila Innis for their support and valuable insights into the project. I gratefully acknowledge the British Columbia (BC) Women’s Hospital and Health Centre and Vancouver Coastal Health for their support in this research. Sincere thanks to the co-investigators Barbara Crocker and Jeannie Dickie for their contributions towards this research. My gratitude to Dr. Susan Whiting for the food frequency questionnaire analyses, Dr. Ruth Mimer for her assistance in statistical analyses, and the BC Biomedical Laboratory for completing the laboratory analyses. I would like to thank Kathleen Lindstrom, the perinatal program manager at Douglas College, and instructors Ana-Maria Orb, Pat Agon-Chen, Pauline Clare, and Bernice Cabana for allowing me to recruit participants from their perinatal classes. Many thanks to the phlebotomists Raj Padda and Maria Kosovic. I want to thank all the participants who volunteered their time and the enthusiasm they brought to the project. Without them, I could not have completed this project. Lastly, I wish to express my thanks to my parents and friends for their continuous encouragement and support. viii CHAPTER 1: INTRODUCTION Vitamin D is a fat-soluble vitamin and also a hormone best known for its role in maintaining blood levels of calcium, by increasing intestinal absorption and reducing urinary calcium excretion (I). It is now widely accepted that vitamin D also has functions that are not related to calcium homeostasis, specifically in cell differentiation and immune function (2). Inadequate vitamin D status has been associated with a wide range of diseases and conditions such as cancer (3), cardiovascular disease (4), and type 1 diabetes (T1D) (5). Humans obtain vitamin D mainly through skin synthesis by the action of ultraviolet (UV) light (6). In the absence of UV light humans are reliant on dietary or supplemental sources of vitamin D. Major natural food sources of vitamin D are limited to fatty fish and organ meats (7). Foods fortified with vitamin D, such as milk and margarine, are available in some countries, for instance, Canada and the United States (US) (8). Circulating 25 hydroxyvitamin D (2501{D) is the best indicator of vitamin D status as it represents vitamin D obtained from both UV skin synthesis and dietary sources (9, 10). The optimal 250HD concentration in pregnancy is not known with certainty. However, it is generally accepted that maintaining a 250HD> 50 nmolfL is desirable for bone outcomes in adults and infants (11, 12) and the Canadian Pediatric Society has recently adopted > 75 nmol/L as “sufficient” for pregnant and lactating women, and infants (13). A number of factors may affect the intensity or effectiveness of UV light on cutaneous synthesis of vitamin D. These factors include environmental conditions such as latitude (14), season (15) and climate (16), as well as use of sunscreen (17), clothing coverage (18), skin color (19), obesity (20), and age (21). Vitamin D is important at all ages, but obtaining adequate vitamin D during pregnancy may be especially important for health of both mother and child. Vitamin D 1 inadequacy during pregnancy has been associated with preeclampsia, the leading cause of maternal morbidity and mortality in Canada (22, 23). Poor vitamin D status during pregnancy may also adversely affect calcium homeostasis and skeletal mineralization of the unborn child. Rickets, which still occurs in Canada, is found almost exclusively in breastfed infants born to vitamin D deficient mothers (24). In addition, a lack of maternal vitamin D may increase risk of Ti D (5) and low bone mineral content (25) later in life. Low vitamin D status during pregnancy, based on low circulating concentrations of 250HD, has been reported in several countries (26, 27). However, there were few studies of pregnant women in Canada; yet there is reason to suspect a high rate of vitamin D insufficiency. Factors that might predispose Canadian women to poor vitamin D status include living at a high latitude, foods naturally rich in vitamin D are not commonly consumed, and for some individuals, darker skin pigmentation. Only 35% of Canadian women of reproductive age achieve the Adequate Intake (Al) for vitamin D of 5 j.tg!d (28, 29), an intake that many experts now consider woefully inadequate (30). For example, the Canadian Pediatric Society has recommended that women receive 50 p.g/d of vitamin D throughout pregnancy, an amount 10 times the current recommendation (13). However, this recommendation is not endorsed by Health Canada, and it is not clear to what extent women are following this advice. Vitamin D recommendations are intensely debated, and are currently under review by the Institute of Medicine with an expected release in fall 2010. The committee will look beyond vitamin D and bone health to consider chronic and nonchronic disease indicators (31). Vitamin D insufficiency increases circulating parathyroid hormone (PTH), which accelerates bone turnover leading to bone loss. PTH concentration is inversely associated with 250HD concentration up to a threshold, above which PTH concentration plateaus at a minimum level. This threshold is often used as a biological criterion to define vitamin D 2 adequacy (32). Although PTH concentrations do not appear to be elevated during pregnancy and lactation, the few studies that examined the relationship between PTH and 250HD have found inconsistent results (33-35). The inconsistency may be a result of a lack of variation in 250HD, not controlling for calcium intake, ethnic differences, or small samples (36). Melanin in skin acts as a natural sunscreen that reduces endogenous vitamin D synthesis. Darker skinned ethnic groups tend to have lower 250HD than lighter skinned ethnic groups living in the same geographical area (37). However, ethnicity is only a proxy measure for skin color, which varies within an ethnic group. Skin color can be quantified using reflectance colorimetry (38). As skin color darkens in response to sun exposure, any measure of darker skin by tanning would therefore tend to have a positive relationship with vitamin D status. Conversely, it would be expected that the darkness of unexposed skin would be negatively related to vitamin D status, as melanin acts as a UV filter (37). Given the widening spectrum of adverse maternal and child outcomes associated with a lack of vitamin D during pregnancy and the paucity of Canadian data the objectives of this research are to determine in a group of Vancouver pregnant women from diverse backgrounds: 1. the prevalence of vitamin D deficiency and insufficiency based on 250HD concentrations; 2. vitamin D supplement use and dietary vitamin D intake; 3. the importance of season, ethnicity and vitamin D intake (including supplements and fortified foods) in determining 250N1) concentrations; 3 4. the association between PTH and 2501{D concentrations and to detenmne if a threshold exists above which further increases in 250HD do not further suppress PTH concentration; 5. the relationship between tanning and natural skin color measured using reflectance colorimetry and 250H]) concentration. 4 CHAPTER 2: LITERATURE REVIEW The first four sections of the literature review include vitamin D metabolism, assessment of vitamin D status, defming vitamin D deficiency and insufficiency using different criteria, and factors affecting 250HD concentration. These are followed by discussions of vitamin D status of pregnant and lactating women in Canada, US, and other countries. Current recommendations for intake of vitamin D for pregnant and lactating women are summarized and consequences of inadequate vitamin D on both mother and infant are discussed. Finally, vitamin D supplementation during pregnancy and lactation and its impact on maternal 250HD concentration and neonatal outcomes is summarized. 2.1 Vitamin D Metabolism There are two major forms of vitamin D: vitamin D2 (ergocalciferol) (Figure 2.1) and vitamin D3 (cholecalciferol) (Figure 2.2). These molecules differ in side-chain structure. Vitamin D2 has an additional C24-methyl group and an extra double-bond between carbons 22 and 23 compared to vitamin D3 (6). Vitamin D2 is produced by the action of ultraviolet light on the fungal sterol, ergosterol. Vitamin D3 originates from the cutaneous sterol, 7-dehydrocholesterol (7-DHC), which is distributed throughout the epidermis and dermis in the skin of animals and humans (6). Upon exposure of skin to UVB portion of sunlight (spectral range 290-320 mn), 7-DHC is converted to previtamin D3 (preD3). PreD3 undergoes a temperature-dependent rearrangement to form the thermally stable vitamin D3 (6). Vitamin D2 and D3 were once believed to have equal bioavailability, but recent studies suggest that vitamin D3 increases serum 250F1D concentrations, the major circulating form of vitamin D, to a greater extent than does vitamin D2 (39, 40). Annas et al (41) reported that peak 250HD concentrations after a 5 large dose of D2 and D3 (1250 tg) were sunilar, but 250HD concentrations decreased more rapidly after D2. Humans obtain vitamin D mainly from exposure to UV light, In the absence of UV light humans are reliant on dietary or supplemental sources of vitamin D. Vitamin D is found naturally in few foods. Major natural food sources of vitamin D are limited to oily fish and animal liver, although small amounts also occur in meat (7). Foods fortified with vitamin D, such as milk, margarine and orange juice are available in some countries, for instance, Canada and the US (8) (Table 2.1). Vitamin D (D2 or D3) from sunlight and dietary sources must be hyciroxylated twice before it is metabolically active (42). Vitamin D, dietary or synthesized, is taken up into the bloodstream where it binds vitamin D binding protein (DBP) for transport. DBP is found in body fluids (43) and various tissues (44), such as skin, kidneys and intestine. The first hydroxylation occurs in the liver, where vitamin D is metabolized to 250HD by the enzyme vitamin D 25-hydroxylase (25-OHase or CYP27A1) (42) (Figure 2.3). In the kidneys, 250HD is further hydroxylated to the biologically active form of vitamin D, I a,25 dihydroxyvitamin D [1 ,25(OH)D1, also known as calcitriol, by the enzyme 1-ct-hydroxylase (1-ctOHase or CYP27B1) (45). 1 ,25(OH)D is the most important metabolite in vitamin D metabolism. This second hydroxylation is tightly regulated by the action of PTH, which stimulates the production of, 1 ,25(OH)Din response to hypocalcemia. More than 30 tissues, including the intestine, bone, and kidney, are known to have receptors for l,25(OH)D (46). In response to low extracellular calcium concentrations, PTH and 1,25(OH)D increase serum calcium concentrations through three separate activities, In the small intestine, l,25(OH)D enhances absorption of calcium and phosphate. Both 1,25(OH)Dand vitamin D receptor are required for optimal intestinal absorption of calcium (47). If dietary calcium is low, 1 ,25(OH)Dhelps regulate 6 serum calcium concentrations by initiating the transfonnation of monocytic stem cells to mature osteoclasts, which are involved in bone resorption (48). Lastly, I ,25(OH)D stimulates renal tubular reabsorption of calcium to increase its serum concentrations (49). It is now widely accepted that vitamin D also has functions that are not related to calcium homeostasis, specifically in cell differentiation and immune function (2). Inadequate vitamin D status has been associated with a wide range of diseases and conditions such as cancer (3), cardiovascular disease (4), and T1D (5). 2.2 Assessment of Vitamin D Status Vitamin D status can be potentially assessed by three biomarkers: serum PTH, l,25(OH)D (Figure 2.4), and 250HD concentrations (Figure 2.5). Serum PTH is inversely associated with circulating 250HD. Vitamin D deficiency results in increased PTH synthesis and secretion. As a person becomes vitamin D-deficient, the efficiency of intestinal calcium absorption is decreased. Once calcium concentration starts to drop, the decrease is recognized by parathyroid glands, which increases the production of PTH. PTH stimulates osteoclast activity leading to bone resorption and increases reabsorption of calcium in the kidneys (48). Factors other than serum 250HD also affect PTH concentration (i.e., physical activity, calcium and phosphorus intake, and plasma phosphate concentration) limiting its usefulness as an indicator of vitamin D status (50). Further, PTH concentration is not a good indicator of vitamin D status because it is inversely associated with serum 250HD concentration up to a threshold, above which it plateaus at a minimum level (32). However, PTH has been used to determine the cut-off values for circulating 250HD to defme vitamin D sufficiency (Section 2.3.1). Circulating 1 ,25(OH)Dconcentration is tightly regulated by PTH, serum calcium and phosphorous, thus it is not a good indicator of vitamin D status. Vitamin D 7 deficiency may be misdiagnosed because physicians incorrectly measure the active form of vitamin D, 1,25(OH)D, to determine status. Indeed, l,25(OH)Dblood concentration remains normal with mild vitamin D deficiency. Concentrations of 1,25(OH)Dmay rise and then fall only under the circumstances of more severe vitamin D deficiency (48). Circulating 250H1) concentration is the best biomarker of vitamin D status because it represents vitamin D from both dietary and endogenous sources (9, 10). It also has a long half-life of about 2 weeks in the circulation and its concentration is not under tight homeostatic regulation (48). 2.3 Defining Vitamin D Deficiency and Insufficiency Circulating 250H1) has been used to defme vitamin D status. Cut-off values for circulating 250HD that defme vitamin D deficiency and insufficiency are generally based on cohort studies, randomized controlled trials, and small metabolic studies. Vitamin D deficiency is defmed as a concentration of below which there is an increased risk of rickets in infants and children and osteomalacia (or “soft” bones) in adults (51). Vitamin D insufficiency is defined as a 250HD concentration not low enough to cause rickets or osteomalacia but may be associated with an increased risk of chronic disease (51). Vitamin D deficiency is well defined as a 2501{D concentration below 25 nmoIJL. Below this concentration the risk of rickets and osteomalacia increase substantially and nearly all people with confirmed cases of these diseases have 250HD concentrations below this cutoff (52). In contrast, there is currently no universally accepted optimal range or cut-off value for 250HD to defme vitamin D insufficiency and cut-offs have ranged from 37.5 to greater than 100 nmoIfL. Part of the problem is that different assays for measuring 250HD are used (53). Different criteria have been used to 8 defme vitamin D insufficiency including PTH suppression, intestinal calcium absorption, bone density, and fracture risk. 2.3.1 Parathyroid hormone suppression Few studies have directly measured the relationship between 250HD and bone resorption, so elevated PTH concentrations have been used as a proxy indicator for bone resorption. PTH is released from parathyroid gland in response to a low serum calcium concentration, which may be caused by a low vitamin D level and impaired calcium absorption. Therefore, PTH is inversely related to serum 250HD. However, this statement may not apply to all race groups. For example, in a study of American white (n 53) and black (n = 52) adolescent girls, a negative relationship between 250HD and PTH was found only in white girls (P = 0.0007), but not blacks (P = 0.30). The authors suggested that if blacks in their cohort had a wider range of 250HD concentrations, an inverse relationship might have been observed as well (54). Excessive secretion of PTH leads to a condition called secondary hyperparathyroidism. Secondary hyperparathyrodism (secondary to vitamin D deficiency) increases bone turnover and is the mechanism by which insufficient vitamin D leads to bone loss (55). It is generally agreed that the lower desirable concentration for 250HD is that which maximally suppresses serum PTH (56); however, others have suggested that maximally suppressing PTH [chronically elevated 1 ,25(OH)D1 may result in prolonged bone resorption and have detrimental effects on bone (49). The 250HD concentration that maximally suppresses PTH has varied by study ranging from 30 (57) to 90 nmolJL (58). For example in one study, secondary hyperparathyroidism was avoided in the elderly only when circulating 250HD concentrations reached 75 or 80 nmol/L, meaning that 250HD has the ability to suppress PTH (< 3.7 pmolJL) at a concentration above 75 nmol!L (59, 9 60). This wide range of estimates may be due to different assays, the influence of calcium intake, population characteristics, and statistical models used to defme the relationship between PTH and 250HD (56). For instance, the relationship between 250HD and PTH appears to be influenced by calcium intake and age. In a study of 39 Italian elderly osteoporotic women with a low calcium intake (< 800 mg!d), a much higher concentration of 250HD was needed ( 110 nmoIJL) to maintain their serum PTH concentrations within the normal range (< 3.7 pmol/L). The high 250HD concentration was achieved by providing them with vitamin D supplements (50-75 jig/d) for 8 months (56). A study conducted among healthy Icelandic adults (n = 944) has found that a high calcium intake (> 1200 mgld) allowed somewhat lower serum 250H1) concentrations for maintaining ideal serum PTH (36). These studies suggest that calcium intake is an important modifier of the PTH and 250HD relationship. 2.3.2 Calcium absorption In addition to the effects of calcium intake on 250HD concentration, there is also an effect of 250HD on calcium absorption. Net calcium absorption (difference between ingested intake and fecal output) increases as calcium intake increases. Net calcium absorption is low at low calcium intake, irrespective of vitamin D status. In addition, it is very difficult to absorb sufficient calcium in the absence of active vitamin D-mediated absorption (61). Therefore, both calcium and vitamin D may be needed to ensure sufficient net absorption of calcium for meeting various body needs. Heaney (62) reported that absorptive efficiency of calcium increased until a plateau level was reached at a serum 250HD concentration of 80 nmol/L In a study of 26 healthy men, calcium absorption fraction following a summer season of extended outdoor activity was found to not have changed in winter. The median serum 250HD concentration dropped from 122 10 nmoIfL in late summer to 74 nmol/L in winter. It appears that 250HD concentration was high enough in winter that calcium absorption was not affected (63). Some studies indicate that calcium absorption varies by 250HD concentrations. An oral 500 mg calcium was given to postmenopausal women whose mean 250HD concentrations were 86.5 (n 24) and 50 nmolfL (n = 24). By measuring the area under the curve of serum calcium profiles, it was found that calcium absorption was 65% higher in the subjects with higher 250HD concentration (86.5 nmoIJL) (64). These results suggest that 250HD concentrations of 75 nmolfL or greater may be necessary to maximize calcium absorption in healthy men and elderly women. However, the relationship between 250HD and calcium absorption may not apply to all age, sex, and race groups. For example, in growing adolescents (n = 105), Weaver et al (54) found no association between calcium absorption and 250HD over a wide range of 250HD concentrations, so that other hormones or factors may be more important determinants of calcium absorption in some groups. 2.3.3 Bone density and facture risk Bone mass or fracture has been used to defme optimal 250HD concentrations. Vitamin D insufficiency is very common in the elderly population and is associated with a high risk of low bone mass which could be related to osteoporosis (21, 65). Some researchers have found that a 250HD concentration below 50 nmol!L is associated with a higher risk of osteomalacia, osteoporosis, fracture and falling (21, 65). In Bangladeshi garment workers (18-36 y; n = 200), a trend of progressive increase in femoral neck and lumbar spine bone mineral density was observed with a serum 250HD concentration greater than 38 nmoIfL, which was found to be the threshold to suppress PTH (65). In the US third National Health and Nutrition Examination Survey (NHANES III, 1988-1994), 11 a significant association was found between 250HD concentrations and total hip bone mineral density in both younger and older white, Mexican American, and black adults. In older whites, this association was positive throughout the reference range (22.5-94 nmol/L) and appeared to plateau in the range of 90-100 nmolfL (66). In studies providing 17.5-20 jig/d of vitamin D3 per day, optimal prevention of both nonvertebral and hip fracture occurred in patients whose baseline concentration of 250HD were less than 42 nmoiIL and whose mean concentration of 250HD then rose to approximately 100 nmoIJL (12). In summary, it is suggested that the cut-off values of 250HD should be at least 50 nmolJL for maximal bone health in general population and 75-100 nmol!L to prevent bone related diseases in elderly individuals (59, 60, 67). Some studies have suggested even higher cutoffs based on cancer and other chronic disease risk prevention (48). When using these cut-off values, issues such as different seasons, subject groups, methodologies, and calcium intake should always be considered. It is important to note that most studies on 250HD have focused on older people and that we have very little data on the optimal 250HD concentration in other populations, especially pregnant and lactating women. Due to a lack of certainty about optimal 250HD concentrations, in the current study a number of cutoffs will be used to defme vitamin D insufficiency. 2.4 Factors Associated with 25-Hydroxyvitamin D Concentration As mentioned in Section 2.1, vitamin D is synthesized by the action of UVB light on 7-DHC, which is distributed throughout the epidemiis and dermis in the skin of animals and humans (6). Vitamin D can be obtained from a few natural food sources, fortified foods, and dietary supplements (7, 8); however, sunlight is the major source of vitamin D when there is sufficient UV light available. A number of factors may affect the 12 intensity or effectiveness of UVB light on cutaneous synthesis of vitamin D. These factors include environmental conditions, skin color, ethnicity, tanning, sun avoidance, sunscreen use, clothing, obesity, diet, and vitamin D fortification. 2.4.1 Environmental conditions: latitude, season and climate The major determinant of 250HD status is exposure of the skin to sunlight. Environmental conditions such as latitude, season and climate all affect dermal vitamin D synthesis. The angle of the sun is acute close to the equator, and dermal vitamin D synthesis through exposure of the skin to sunlight would be expected to occur throughout the year. At higher latitudes, sunlight reaches the earth at a more oblique angle; and therefore results in UV rays traveling a greater distance through atmosphere (14, 68). As atmospheric absorption of UV radiation increases, the potential for vitamin D synthesis reduces. This effect becomes more pronounced as latitude increases when there is a shorter period of the year during which UVB radiation is sufficient to produce vitamin D, particularly in winter months (14, 68). A seasonal variation in 250HD concentrations is well described. Many studies conducted in different countries, for example Canada; northern Europe, New Zealand, Australia, and the US have shown that vitamin D deficiency or insufficiency is more prevalent during winter than summer (15, 16, 20, 69-73). As indicated in the 2007-2009 Canadian Health Measures Survey (CHMS) (6-79 y; n = 5306), 250HD concentrations tended to be lower among people whose blood was drawn in November-March [64.1(60.3, 679) nmol!L] rather than in April-October [70.0 (65.6, 74.4) nmoJfLJ. The percentage with optimal 2501{D concentrations (> 75 mnol!L) was significantly higher in summer months (39%) than in winter months (30%) (74). It is interesting to note that a much greater seasonal effect was found among New Zealand children and adolescents (n 1583) who participated in the 2002 National Children’s 13 Nutrition Survey (Figure 2.6) than that reported in the CHMS. There was a mean difference of 15 (9, 21) nmoIfL between winter and summer in the New Zealand survey compared to only 6 nmoIfL in the CHMS (71). In addition, cold weather during the winter months at higher latitudes requires more clothing thus there is less UV exposure (16). Other environmental conditions with the potential to negatively affect dermal vitamin D synthesis include air pollution and urban living associated with an indoor lifestyle (16, 69, 75-78). 2.4.2 Skin color and ethnicity Dermal vitamin D synthesis is greatly influenced by skin pigmentation. Melanin in skin acts as a natural sunscreen and it absorbs UV light and therefore prevents the UV photons from entering skin cells to covert 7-DHC into preD3 (19). To obtain an equivalent amount of vitamin D, a darker skinned individual (high melanin content) requires a longer period of UV exposure than a fairer skinned individual (low melanin content) because vitamin D synthesis is less efficient among darker skinned people (19). Darker skinned ethnic groups tend to have lower vitamin D status than fairer skinned ethnic groups living in the same geographical area. In the CHMS, white racial background tended to be associated with higher 250HD concentrations compared to non-whites [71.2 (68.8, 73.7) and 52.3 (49.1, 55.5) nmol/L; P < 0.05] (74). Vitamin D deficiency and insufficiency is more prevalent in Hispanic and African American groups than in Caucasian groups (72). In a UK inner-city multicultural population (n = 830), one in eight Caucasians, one in four black Afro-Caribbeans, and one in three Asians were deficient in vitamin D (< 25 nmoiJL) suggesting that lighter skinned people were less likely to have poor vitamin D status compared with darker skinned people (79). A cross-sectional survey (National Children’s Nutrition Survey 2002) in New Zealand 14 children aged 5-14 concluded that the risk of vitamin D insufficiency (< 37.5 nmol/L) doubled in Mäori and Pacific children, who tended to have darker skin, compared with New Zealand Europeans (71). Data from the NHANES III showed that white men and women (n = 15390) had higher mean 250HD concentrations (83 and 76 nmolJL) than Hispanic men and women (68.3 and 56.7 nmoL/L; P <0.0001) and than black men and women (52.2 and 45.3 nmolIL; P < 0.0001) (80). In the NHANES 2001-2004, Non-Hispanic black or Mexican-American children aged 1 to 21 were more likely to be 250HD deficient (<37.5 nmol!L) compared with non-Hispanic white children (81). Vitamin D status of pregnant women and women of reproductive age is also related to ethnicity. Serum 250HD concentrations were measured at — 21St week of gestation in black (n = 200) and white pregnant women (n 200) residing in the northern US (40°N). Although both ethnic groups had a high prevalence of vitamin D insufficiency (37.5-80 mnolJL), the vast majority of black women were vitamin D deficient (< 37.5 nmolJL), while almost none of white women were deficient in vitamin D (26). In a study conducted in a multinational refugee population in New Zealand (37°S), 78% of women of reproductive age (n = 192) had poor vitamin D status (< 50 nmol/L) (82). Vitamin D status is associated with ethnicity presumably due to skin color; however, ethnicity is only a proxy measure of skin color. Skin color varies greatly within ethnic groups. In one study, healthy adults in Boston with skin type II (Caucasian skin), III, IV, and V (black skin) were administered 0.75 minimal erythema dose (the minimum amount of UVB that produces redness 24 hours after exposure) in each session for 36 sessions. UVB irradiation greatly increased 250HD concentrations in all skin types. The percent increases were 210 ± 53%, 187 ± 64%, 125 ± 55%, and 40% for skin type II to V, respectively (83). The results suggest that the conversion of 7-DHC to preD3 in type II skin was about 5-fold more efficient than the highly pigmented type V skin. Therefore, 15 Caucasians were more likely than non-Caucasians to obtain sufficient vitamin D after a relatively short period of sun exposure (83). Skin color can be quantified using reflectance colorimetry. Data is reported using the Commission Internationale de l’Eclairage L*a*b* system. A higher L* value represents lighter (or brighter) skin that contains less UV absorbing melanin (37). One may expect that as the color of unexposed skin gets lighter, 250HD concentrations should be higher. Conversely, as skin color darkens with sun exposure, a measure of tanning would be positively associated with 250HD concentrations. In a group of Pacific Islanders (n = 82) and Europeans (n = 239) living in New Zealand (47°S), the association between tanning (sun-induced) and natural (constitutive) skin color and 25OH]) concentrations was determined. It was concluded that tanning skin color was an important detenninant of 250HD. In contrast, natural skin color was not associated with plasma 250HD concentration. This might be due to a lower proportion of darker skinned people included in their study (37). In another study, the unexposed skin L* value and baseline 25OHD concentrations were positively associated. The authors concluded that a typical northern European person would have an L* value for unexposed skin of about 70; an African American would have an L* value of about 50; and a sub-Saharan African would have an L* value of about 35. In order to increase 250HD concentrations by 30 nmol!L, the typical northern European, African American, and the sub-Saharan African would need to receive 39, 55, and 78 mJ/cm2of UVB, 3 times a week for 4 weeks, respectively (73). 2.4.3 Tanning, sun avoidance, sunscreen and clothing The popularity of indoor tanning for cosmetic reasons is growing in North America (84). Tanning has been associated with higher 250111) concentrations. In a 16 cross-sectional study, regular tanning bed users had serum 250HD concentrations 90% higher than nontanners (115.5 ± 8 and 60.3 ± 3 nmol/L, respectively) (85). However, it is suggested that tanning is not a safe way to increase vitamin D levels (86). Tanners have higher risks of acute sunburn and skin cancer compared with non-tanners (87). It appears that people from different cultures have different attitudes toward sunlight exposure. In a survey conducted among middle-age and elderly Chinese women living in Hong Kong (n = 547), 62% of the participants did not like going in the sun, 44% used a parasol to shade themselves from the sun, and the youngest age group of 50-54 y used sunscreen most frequently (75). One possible reason for sun avoidance is the current cultural trend prevalent among many Asian female populations to have paler or fairer skin. Many of these Asian research participants were unaware of the relationship between vitamin D inadequacy and sun protection (75, 88). There is no thta on how prevalent this practice is among Asian women living in Canada; however in a survey of Californian Asian Americans, the adoption of Western culture may be associated with attitudes and behaviours promoting sun exposure, for example the belief that having a tan is attractive, negative attitudes toward use of sunscreen and sun protective clothing (89). Sunscreens are used to prevent sunburn, skin aging, and reduce the risk of skin cancer (17). Daily sunscreen use protects individuals from developing skin cancer as it results in 40% decease in squamous cell carcinoma tumors (90). Sunscreen use has been shown to greatly suppress cutaneous vitamin D synthesis under very strictly controlled conditions; however, their normal usage does not generally result in vitamin D deficiency (16). This may be because, in practice, sunscreens are not applied at the concentration that provides the tested level of protection and they are not used to cover all exposed skin (16, 9 1-93). For example, in southeast Queensland (27°S), Australia, regular winter sunscreen users (n = 31), who had normal weight and engaged in outdoor activities, had a 17 mean (95% CI) serum 250HD concentration of 60.3 (50.1, 78.8) nmol/L(16). Clothing reduces photosynthesis of vitamin D. Direct transmission of UVB is reduced the most by black wool and the least by white cotton. Cultural practices such as complete clothing cover effectively minimize sun exposure (18). Low vitamin D status among veiled women has been reported in several studies. For instance, in a group of veiled women living in Melbourne, Australia (37°S; n = 82), 80% were at high risk of vitamin D deficiency with serum 250HD concentrations below 22.5 nmol/L (27). Indeed, some of the lowest 250HD concentrations have been reported among veiled Arab women living in the Middle East where there is sufficient UV for vitamin D synthesis year round (94). 2.4.4 Obesity Overweight and obesity appears to be associated with lower vitamin D status in several populations, such as Norwegian patients seeking weight-loss treatment (95), postmenopausal women in UK and New Zealand (20, 96), and obese children (97, 98). In a US study, vitamin D status was assessed in obese people and healthy non-obese controls [Body Mass Index (BMI) <30 kg/rn2]matched for age, gender, ethnicity, and season of vitamin D measurement. A higher proportion of obese people had vitamin D deficiency (<50 nmol/L) and insufficiency (50-75 nmoIJL) compared with non-obese people. The differences were unchanged after controlling for sunlight exposure and dietary intakes of calcium and vitamin D (99). A lower concentration of 250HD may be related to decreased exposure to sunlight in obese subjects due to limited mobility or excessive deposition of vitamin D in the adipose tissue (20, 77, 100). When 25OHD concentrations were measured in black and white pregnant women and in their neonates’ cord blood, pre-pregnant obese women 18 (BMI 30 kg/rn2) had a lower mean 250HD concentration than that of lean women (BMI <25 kg!m2): 55.9 (48.7, 64.2) ef. 62.8 (55.0, 70.4) nmol!L, respectively (P < 0.05). Obese women also had a higher prevalence of vitamin D deficiency (< 50 nmol!L) compared with lean women (61 and 36%, respectively; P < 0.01). There was a dose-response trend between pre-pregnancy BMI and vitamin D deficiency. Additionally, neonates born to obese mothers had poorer vitamin D status than those of lean mothers (101). One study has suggested that obesity may play a less important role in explaining variation in circulating 250HD in black women compared with white women. Among non-Hispanic whites (n 3567) and non-Hispanic blacks (n = 2475), the negative relationship between serum 250HD and % body fat was stronger in whites than in blacks of the same age. The authors stated that the reason for the weaker relationship between body fat and 250HD in blacks was not clear; however, it is possible that body fat had a lower impact in blacks because there was reduced synthesis of vitamin D in the skin due to the presence of more melanin (102). 2.4.5 Diet and fortification Dietary vitamin D intake may be a more important determinant of circulating 250HD concentrations in countries with high fatty fish consumption such as Japan or in countries with vitamin D fortification such as the US and Canada. When the vitamin D status of women aged 6 1-86 living in central Sweden (60°N) during winter was measured, serum 25OH1) concentrations increased by 10.2 nmolJL with each additional 130 g fatty fish per wk (i.e., salmon and herring), by 6.2 mnolJL with the daily intake of 300 g vitamin D-fortified dairy products, and by 11.0 nmol!L with regular use of vitamin D supplements (103). Jn Canada and the US, vitamin D fortification is regulated. It is mandatory to 19 fortify cow’s milk (2.5 ig/250 mL) and margarine (13.3 .tg!l00g) with vitamin D in Canada (8). Goat’s milk, fortified plant based beverages (i.e., fortified soy beverages), and some calcium-fortified orange juices are permitted to be fortified with vitamin D. Cheese and yoghurt can be made with vitamin D-fortified milk; however, the final product does not contain as much vitamin D as fluid milk alone. Fluid milk is the major dietary source of vitamin D because it is a commonly-consumed food in Canada (104). In the US, foods that are commonly fortified with vitamin D include dairy products (i.e., milk, yoghurt, and cheese slices), calcium-fortified orange juice, margarine and other spreads, and breakfast cereals (105). Fortification in Canada and the US may explain, in part, the higher 250HD concentrations compared to countries without fortification (74). In Canada, it is reconunended that prenatal supplements contain 10 ig/d of vitamin D3 (13). Vitamin D supplementation is associated with higher 250HD concentrations and this is covered in Section 2.8. 2.5 Vitamin D Status of Pregnant and Lactating Women Worldwide there have been several studies that have described the vitamin status of pregnant and lactating women. Surprisingly there are few studies of Canadian pregnant and lactating women. 2.5.1 Canada There have been two studies that have reported poor vitamin D status among Aboriginal pregnant and lactating women. Among Canadian mothers living in the Inuvik zone of the Northwest Territories (68°N), both native (51 Inuits and 37 native Indians) and non-native (33 Caucasians) pregnant women were vitamin D insufficient despite most women talcing a supplement containing vitamin D (10 i.g/d). At delivery, the mean 20 plasma 250HD concentration was lower among native mothers (50.1 ± 19.3 nmoIJL) than non-native mothers (59.8 ± 29.4 nmol!L) (106). In native Canadian Cree in northern Manitoba (53°N) (n = 80), lactating women were found to be severely deficient in vitamin D with a mean serum 250H1) concentration of 19.8 nmoli’L. The authors attributed this to limited endogenous vitamin D synthesis (high latitude and limited sun exposure), low consumption of fortified milk, and limited use of vitamin D supplements (107). In a study of predominately white pregnant women from Edmonton (53°N) (n = 83), about 23% were vitamin D deficient and 53% were insufficient, based on cutoffs of < 40 nmoL/L and 40-80 nmoIfL, respectively (108). There are two studies of predominately white pregnant women living in the province of Newfoundland and Labrador (47°N and 52°N). In the first study, pregnant women (n = 50, 98% Caucasians) were randomly selected from 79 census consolidated subdivisions across Newfoundland and Labrador. About 2% were vitamin D deficient (< 25 nmolfL) and 78% were vitamin D insufficient (25-75 nmol/L); however, their 250HD concentrations were not significantly different in winter (51.9 nmoIJL) and summer (61.1 nmol/L) (109). In the second study, women were recruited from the Avalon Peninsula of Newfoundland (47°N). Their mean 250HD concentrations were 52.1 nmol!L and 68.6 nmol!L in the winter (n = 304) and summer (n 289), respectively. Vitamin D deficiency (< 25 nmol!L) was more prevalent in the winter than in the summer (6.6 and 1.7%, respectively). Vitamin D insufficiency (25-75 nmolfL) was also more prevalent in the winter than summer (83% and 62%, respectively) (110). Because there are few studies on the vitamin D status of pregnant and lactating Canadian women, it is reasonable to consider the status of non-pregnant women of reproductive age. There have been a number of studies, mostly small convenience samples that have described the vitamin D status of non-pregnant Canadian women. 21 However, data from the 2007-2009 CHMS provide the most recent population based estimates of 250HD in Canadian population. Females of childbearing age (20-3 9 y; n = 650) had a mean (95% CI) 250HD concentration of 69.5 (65.8, 73.2) nmol!L. About 3% of women were considered vitamin D deficient (< 27.5 nmol./L). About 10% had concentrations below 37.5 nmoL/L and 64% had concentrations below 75 nmol/L (74). As indicated in this survey, lower vitamin D status was associated with winter season, non-white racial backgrounds, and less frequent intake of fortified milk. Women 20-39 y had a significantly higher mean 250HD concentration in summer than winter: 74.3 (67.5, 81.1) and 64.2 (59.7, 68.8) mnoifL, respectively. The mean (95% CI) 250HD concentration was significantly lower among non-white women [48.9 (46.5, 51.2) nmoL/L] than white women [75.5 (71.5, 79.6) nmoL/L]. The results indicated that only a small number of Canadian women of reproductive age had 250HD concentrations below 37.5 nmoIJL (74). 2.5.2 United States ofAmerica Vitamin D status of American pregnant women from different ethnic backgrounds has been reported in a few studies. In Pittsburgh, northern US (40°N), vitamin 0 status was determined among 200 white women and 200 black women. At term, the mean (95% CI) 250HD concentration of black women [49.4 (46.1, 52.9) nmolJLj was significantly lower than that of white women [80.4 (76.0, 85.1) nmoIfL]. About one half of women in both groups were insufficient in vitamin D (37.5—80 nmoIJL); however, only 5% of white women were vitamin D deficient (< 37.5 nmol/L) whereas 29.2% of black women were deficient. The results were similar at 4th - 21St week of gestation. The mean 250F1D concentrations of black and white women were 40.2 (37.9, 42.7) and 73.1(69.4, 76.9) nmol/L, respectively. The results suggest that white and black 22 pregnant women living in the northern US were at high risk of vitamin D insufficiency, even when over 90% of women used prenatal vitamins (10 jig/d) (26). In Baltimore, Maryland (39°N), serum 250HD concentration was measured in pregnant African American adolescents during the second trimester (n = 44) or third trimester (n = 36) of pregnancy. The mean 250HD concentrations were not significantly different between the second and third trimester (52.4 ± 20.5 and 56.3 ± 19.8 nmoVL, respectively). About 21% and 46% were vitamin D deficient (< 37.5 nmoiJL) and insufficient (< 50 nmoIfL), respectively (11 1). As for Canada, the status of non-pregnant women of reproductive age is considered because studies on the vitamin D status of pregnant and lactating American women are limited. In the NHANES III, the prevalence and determinants of hypovitaminosis D ( 37.5 nmoIIL) among non-pregnant African American (n = 1546) and white women (n = 1426) of reproductive age (15-49 y) were examined. The mean serum 250HD concentrations were 44.2 ± 1.1 nmol!L and 82.5 ± 1.5 nmoIfL among African American women and white women, respectively. Nearly one-half (42.4%) of African American women had a serum 250HD concentration S 37.5 nmol!L, whereas only 4.2% of whites were below this cut-off concentration. A higher proportion (12.2%) of African American women had 250HD concentrations <25 nmol/L than that of white women (0.5%). Vitamin D inadequacy described in this study was associated with consumption of milk or breakfast cereal less than 3 times a week, no use of vitamin D supplements, winter season, urban residence, and higher BMI. The findings suggest that many African American women in the US could enter pregnancy with a serum 250HD concentration 37.5 nmol!L (112). 23 2.5.3 Other countries Studies from different parts of the world have identified a high prevalence of vitamin D deficiency and insufficiency among pregnant and lactating women, including the United Kingdom (113-115), Europe (116-124), Australia (27, 33, 125), New Zealand (126), Asia (127-130), Middle East (94, 131-137), and Africa (138) (Table 2.2). Studies included in Table 2.2 were conducted in cities with various latitudes ranging from 11 °N (Maiduguri) to 59°N (Oslo); however, most studies were conducted in cities with higher latitudes. Although pregnant and lactating women residing in higher latitudes usually have a higher risk for vitamin D inadequacy, living in a lower-latitude city (for example, Riyadh, 24°N) does not guarantee vitamin D sufficiency (94). Low levels of vitamin D have been reported in regions with ample sunshine. For instance, new mothers in New Delhi were found to have 250HD concentrations around 25 mnolJL (130). As evidenced in these studies, vitamin D status is generally better in summer than in winter (117-119). In addition to latitude and season, cultural practices such as complete clothing cover also affect vitamin D status of pregnant and lactating women. Veiled pregnant women and orthodox Jewish mothers had low 250HD concentrations of 14 nmolfL and 34 nmolJL, respectively (27, 134). In studies comparing vitamin D status between westerns and non-westerns, women of non-western background had significantly lower 25OHD concentrations than western women (120, 122, 123). Overall, it would appear that the vitamin D status of pregnant women in many countries, including Canada, is suboptimal. However, It can be difficult to compare 250HD concentrations between studies due to different assays, population characteristics, seasons, and latitude (139). In Canada, a few studies have reported the 250HD status of pregnant and lactating women residing in the Inuvik zone, Manitoba, Edmonton, and Newfoundland and Labrador. Vitamin D deficiency and insufficiency was common in 24 these women and in general low vitamin D status was more prevalent in winter than summer. Compared to the vitamin D status of Canadian women of reproductive age reported in CHMS, it seems that pregnant and lactating women had much lower 250H1) concentrations and this may be due to small convenience samples of these studies and the locations in which they were conducted. 2.6 Current Recommendations for Intake of Vitamin D for Pregnant and Lactating Women Current Canadian recommendations for intake of vitamin D for pregnant and lactating women vary up to 10-fold. Recommendations for vitamin D from various national and international governments and organizations are given in Table 2.3. The following review will mainly discuss the recommendations and current vitamin D intakes among pregnant, lactating women, and women of reproductive age in North America. 2.6.1 Nutrient reference values Intake reference values for vitamin D are provided in the Dietary Reference Intakes (DRIs) developed by the Food and Nutrition Board (FNB) at the Institute of Medicine of The National Academies. DRIs are reference values used to plan and assess nutrient intakes of healthy people. The values vary for age and gender. These DRIs are used by Health Canada in making recommendations for Canadians. For vitamin D, an Al is established because evidence is insufficient to develop a Recommended Dietary Allowance (29). The current Al for vitamin D for pregnancy and lactation is 5 j.tg/d, which is the same as for all other gender and life-stages 50 y (29). The Institute of Medicine concluded that vitamin D requirements for pregnant and lactating women are not greater than for women who are not. The Al was derived primarily from a study of 59 25 women (25—3 5 y) living in Nebraska. A daily dietary vitamin D intake of 3.3-3.4 jig was sufficient to maintain serum 250HD concentrations greater than 30 nmol/L in most of the women in the winter months (140). The FNB rounded this down to 2.5 jig/d and then doubled it for an Al of 5 jig!d. The World Health Organization (WHO) also recommends 5 jig!d for pregnant and lactating women (141). Interestingly, both organizations indicate that vitamin D supplementation during pregnancy and lactation probably should not be discouraged. Many experts now consider the A! of 5 jig!d woefully inadequate (30). The vitamin D recommendations are being reviewed by the Institute of Medicine and it is possible that recommendations will be increased for most age groups. The Canadian Pediatric Society has recently recommended that pregnant wOmen should receive 50 jig/d of vitamin D, an amount 10 times the current recommendation and equal to the current Tolerable Upper Level (Ut) of intake; however, this recommendation is not approved by Health Canada, and it is not known to what extent women and their health professionals are following this advice (13). The European Union (EU), United Kingdom Committee on the Medical Aspects of Food Policy (COMA), and the UK Department of Health all recommend an intake of 10 jig/d (142-144). The COMA further recommends daily supplementation of 10 jig/d of vitamin D for pregnant and lactating women (144). In Australia and New Zealand, the National Health and Medical Research Council (NHMRC) recommends a daily vitamin D intake of 5 j.tg/d. It also suggests that a supplemental intake of 10 jig/d would not be excessive (145). 2.6.2 Tolerable upper level of intake and toxicity The Ut by defmition poses a very low risk of adverse health effects. For vitamin D, the Ut for healthy adults is 50 jig, an intake that does not lead to hypercalcemia (29). 26 The IJL for pregnancy and lactation is set at the same level as for non-pregnant women because no adverse effects were found among pregnant women who received vitamin D supplement of 25-50 jtg/d (146, 147). Most recent clmical trials have suggested a much higher Ut In a review of risk assessment for vitamin D, a revised vitamin D UL of 250 jigld (10,000 IU) was recommended because foods (unfortified and fortified) and most dietary supplements, combined, do not contribute to a total exposure anywhere near this intake (148, 149). Ordinary dietary sources usually provide about 2.5 jig!d of vitamin D, but can be as high as 5 to 10 jig with the use of fortified foods (8). One serving of salmon (75 g), which is one of the most significant sources of vitamin D, provides about 15 jig (150). Many vitamin D containing dietary supplements for adults provide 5-10 jig!d, when used according to the label instructions (149). Therefore, an intake of 250 j.tg!d would be unlikely to reach. A vitamin D intake up to 2500 jig for a short time or periodically has shown no adverse effects (151, 152). Although large doses of UVB may result in the synthesis of large amounts of previtamin D3, much of this product is degraded into inert photoproducts. Therefore, excessive exposure to sunlight does not lead to vitamin D intoxication (153). 2.6.3 Vitamin D intake from food sources and supplements This section will summarize studies on vitamin D intake of pregnant and lactating women in Canada and the US. Since there are few studies on the vitamin D intake in these groups, it is reasonable to consider the intake of non-pregnant women of reproductive age. In the Inuvik zone in Canada, the daily mean vitamin D intakes of native pregnant women (n = 88), including Inuits and Indians, with and without supplements were 8.1 ± 5.5 and 3.4 ± 2.5 jig, respectively. The total vitamin D intake of Caucasians 27 (13.2 ± 5.9 j.igld) was significantly higher than that of either Inuits (8.2 ± 5.0 tg/d) or Indians (7.9 ± 6.0 tg/d) (106). For Canadians of various age and sex groups, the 2004 Canadian Community Health Survey Cycle 2.2 (CCHS2.2) reported an average vitamin D intake of 5.1 to 7.3 ig/d from food sources, mostly fortified milk. Females (9-70 y) had lower intakes than males (28). In a group of non-pregnant women of reproductive age (25-50 y) residing in Manitoba, about 35% did not meet the AT of 5 gg!d. In this study, ethnicity did not affect total vitamin D intake from both food sources and supplements: 10.6 ± 12.2 (urban Aboriginals, n = 129), 11.3 ± 15.4 (rural Aboriginals, n = 17), and 8.6 ± 8.4 jig/d (white, n = 87). The main food sources of vitamin D for all ethnic groups were found to be milk and margarine. Rurul Aboriginal women consumed all of their dietary vitamin D from food sources (154). In a study conducted among low income minority pregnant women in New Jersey the total vitamin D (supplements and diet) intake was 10.3 ± 0.1 jig/d (4.8 ± 0.1 from diet and 5.5 tg/d from supplements) (155). Vitamin D intakes were also assessed in 1543 pregnant women in Project Viva, a large US pregnancy cohort. Their mean total vitamin D intakes during the 1st and 2nd trimester of pregnancy were 12.6 ± 5.3 and 15.1 ± 4.7 jig/d, respectively (156). It appears that their intakes were higher than Americans as a whole. Data from the NHANES conducted in 1988-1994 and 1999-2000 showed that the mean intakes of vitamin D from all sources were low (5-10 rig) for all of the age and gender-specific ethnic groupings (8, 157, 158). Females generally had lower intakes of vitamin D than males and total intake was lowest in females aged 14-30 (8). Among white, African American, and Mexican American non-pregnant women of reproductive age (19-50 y) in the NHANES 1999-2000, the usual vitamin D intake from food alone was 4.3 ± 0.2, 3.7 ± 0.2, and 4.0 ± 0.3 jig/d, respectively. Vitamin D intakes from both food and supplements for white, African American, and Mexican American women were: 28 7.8 ± 0.4, 6.1 ± 0.4, and 5.7 ± 0.4 ig/d, respectively (157). The use of dietary supplements was associated with a 2-3 jig increase in daily vitamin D intake (8). In the Continuing Survey of Food Intakes by Individuals 1994-1996, 1998, the mean vitamin D intake from food alone was around 3.5 j.tg/d for females 19-30 (n = 760) and about 75% of these women did not meet the Al (159). 2.7 Consequences of Inadequate Vitamin D during Pregnancy and Lactation There is good evidence that vitamin D inadequacy during pregnancy and lactation increases the risk of rickets and may increase the risk of osteoporosis later in life. Because receptors for 1 ,25(OH)Dare found in many tissues, a lack of vitamin D may be associated with other diseases. Although the strength of evidence is not as strong as for rickets, associations between inadequate vitamin D and an increased risk of diabetes, cancers, and multiple sclerosis have been reported (160). Vitamin D deficiency or insufficiency is undesirable at any life stage, but consequences of inadequate vitamin D may be more severe during times of high calcium demand such as pregnancy, lactation, and infancy. Therefore, adequate maternal vitamin D levels are required to ensure fetal bone health and general health of mother and child (161-164). 2.7.1 Vitamin D metabolism during pregnancy and lactation Fetal need for calcium increases throughout pregnancy. The total calcium content of the average 3500 g infants is approximately 28 g (165). The accretion rates of calcium in fetal skeleton increase from 2-3 mg/d in the first trimester to 250 mgld near term (166). To accommodate these higher calcium needs, the rate or efficiency of intestinal calcium absorption doubles, renal excretion of calcium decreases, and resorption of calcium from the maternal skeleton increases during pregnancy (167). Calcium and bone metabolism 29 depend on vitamin D sufficiency, so that adequate vitamin D would seem to be especially critical during pregnancy and lactation. However, maternal adaptations during pregnancy provide required calcium relatively independently of vitamin D. It is only after birth that vitamin D sufficiency becomes important, at least with respect to calcium metabolism and skeletal health (168). 1 ,25(OH)Dis synthesized mainly by the decidual cells of the placenta. During pregnancy, serum concentrations of 1 ,25(OH)D are elevated and with appropriate calcium intake, calcium absorption is increased enough to compensate for the physiologic mechanism of calcium loss or transference to the fetus (169). Although PTH concentrations do not appear to be elevated during pregnancy, the few studies that examined the relationship between PTH and 250HD have found inconsistent results (33-35). The inconsistency may be due to a lack of variation in 250HD, not controlling for calcium intake, ethnic differences, or small samples (36). During pregnancy, maternal concentrations of 250HD correlate well with dietary vitamin D intake; however, few studies have addressed whether the optimal concentration of 250HD for general population applies to pregnant women (169). 2SOHD is believed to cross the placenta and cord blood 250HD concentrations are about 80% of maternal concentrations (170, 171). The fetus depends entirely on the mother’s supply of 250HD (169). The passage of 250HD from mother to fetus could reduce maternal concentrations, especially if the mother is deficient in vitamin D. Therefore, for neonates to be born with normal 250HD concentration, it is essential that their mothers are sufficient in vitamin D during pregnancy (168). After birth, total and ionized calcium concentrations progressively decrease, so that by the first few days of life calcium concentrations are often lower than those found in older infants and children. Calcium concentrations usually return to normal by 5-10 30 days of age (169). In response to the decrease in serum calcium, serum PTH concentrations tend to be low in cord blood but increase within the first 2 days of life. Decreased calcium and increased PTH secretion induce synthesis of 1 ,25(OH)D after birth; however, in vitamin D adequate infants, serum concentrations of 250HD are a rate-limiting factor in the synthesis of 1 ,25(OH)D(169). On average, maternal calcium loss is 4 times higher in lactating than pregnant women because lactation can require 150-300 mg/d of calcium per kilogram of infant’s body weight. Vitamin D passes readily into breast milk, 250HD passes very poorly, and 1 ,25(OH)Ddoes not appear to pass at all. 1,25(OH)D concentrations fall rapidly after pregnancy and are nonnal during lactation. Breast milk should only account for a small loss of 250HD (168). 2.7.2 Musculo-skeletal health of mothers The effect of inadequate vitamin D on maternal bone health is controversial. Although it was indicated in a recent review that maternal adaptations during pregnancy provide sufficient calcium independently of vitamin D, human data is limited (168). Vitamin D deficiency may lead to high bone turnover, bone loss, and osteomalacia (32, 172). In rural North India, 74% of the pregnant women (n = 139) in their second trimester were deficient in vitamin D (25OHD < 50 nmol/L) and biochemical osteomalacia, defmed as heat-stable placental alkaline phosphatase > 240 U/l, was present in 43% of women, but no clinical osteomalacia was found (173). Chronic lower back pain associated with osteomalacia may be related to vitamin D deficiency. Lower back pain is commonly reported during pregnancy because pregnant women suffer from stress on the lower back due to conformational change, additional weight, and extra demands on maternal calcium metabolism. A study conducted in Saudi Arabia concluded that 85% of the female patients (n = 324) attending spinal and internal 31 medicine clinics were deficient in vitamin D (2501{D <22.5 nmoIJL) before vitamin D supplementation. After three months of supplement use (125 or 250 jig/d), 95% of the patients had normal serum 250HD concentrations (> 22 nmoIfL) and reported disappearance of the back pain (174). Severe vitamin D deficiency causes modest hypocalcemia and secondary hyperparathyroidism in nonpregnant adults, but no studies have reported whether these conditions are more or less severe during pregnancy (168). 2.7.3 Non-skeletal health of mothers Vitamin D inadequacy is not only related to maternal skeletal health, but it is also associated with non-skeletal health, such as preeclampsia and gestational diabetes. 2.7.3.1 Preeclampsia Preeclampsia is a pregnancy-specific syndrome characterized by pregnancy-induced hypertension and proteinuria. It affects 3-4% of pregnancies and it is the leading cause of maternal and fetal morbidity and mortality (175). High maternal 250HD concentrations appear to protect against preeclampsia. A cohort study showed that serum 250H1) concentrations in early pregnancy were lower in women who subsequently developed preeclampsia [45.4 (38.6, 53.4) nmoIlL; n = 40] compared with the controls [53.1 (47.1, 59.9) nmoiJL; n = 216] (P < 0.01). This study also found that the risk of preeclampsia (adjusted odds ratio 2.4; 95% CI 1.1-5.4) doubled when 250HD concentration was lowered by 50 nmoIJL (22). Several studies have shown that it is important for infants to have sufficient vitamin D in early stage of their life to reduce the risk of preelampsia in the future. In the Nothern Finland Birth Cohort 1966, 68 (2.3%) (n = 2969) women, who had preeclampsia in their first pregnancy, were found to be deficient in vitamin D in their early life. There was a 50% reduction in the risk of 32 preeclampsia among women who had received vitamin D supplementation regularly during the first year of life compared to those who had received supplementation irregularly or not at all (176). In addition, Bodnar et al (22) indicated that neonates born to preeclampsia mothers were twice as likely as control neonates to have poor vitamin D status (250110 <37.5 nmolIL). Most recently in a cohort study of Norwegian mothers and children (n = 23423), 5.4% developed preeclampsia. A total vitamin D intake of 15-20 j.tg/d was associated with a 25% reduction in the risk of preeclampsia compared with an intake less than 5 .tgld. Considering only the vitamin D intake from supplements, a 27% risk reduction was found for women taking 10-15 jig/d as compared with no supplements. Intake of vitamin D from diet alone was not related to the occurrence of preeclampsia. However, the authors claimed that because vitamin D intake is highly related to n-3 fatty acid intake in the Norwegian diet, it was difficult to distinguish the separate effects of these nutrients (177). These fmdings have shown a protective effect of vitamin D on preeclampsia development; however, more studies are needed to provide firm evidence for a causal association between vitamin D and preeclampsia risk (176). 2.7.3.2 Gestational diabetes In several studies, vitamin D deficiency was associated with an increased risk of gestational diabetes (GD). Although the mechanism is not well defmed, vitamin D deficiency was related to insulin resistance, impaired insulin secretion, and n-cell dysfunction (178, 179). In a group of Australian pregnant women (n 307), maternal plasma 250HD concentrations at 16th week of gestation were negatively correlated with fasting plasma glucose and fasting insulin after adjusting for age, BMI and ethnicity (180). In a nested case-control study, maternal plasma 250H1) concentrations were significantly lower among women who developed GD than controls (62.3 ± 20.5 and 74.3 ± 23.5 33 nmol!L, respectively) after adjusting for BMI. Vitamin D deficiency (< 50 nmolfL) was associated with a 2.66-fold increased risk of GD. Additionally, each 12.5 nmolfL decrease in 25011D concentration was related to a 1.29-fold increase in GD risk (181). In contrast, in a study of South Indian pregnant women (n = 559), such relationship was not found. In their study, the incidence of GD was the same in women with and without hypovitaminosis D (< 50 nmoifL). There were no associations between 250HD concentrations and maternal age, height, BMI or fat mass either (182). 2.7.4 Musculo-skeletal health of infants Vitamin D deficiency during pregnancy has shown negative effects on calcium homeostasis and skeletal mineralization of the unborn child, for example, the occurrence of congenital rickets and low bone mineral content (183-185). Maternal 250HD concentrations in late pregnancy were associated with children’s knee-heel length, which measures intrauterine long bone growth at birth (33), and bone mineral density (25). In a longitudinal study in UK, 198 children were followed up at 9 y. About 18% and 31% of mothers had deficient (< 27.5 nmoIIL) and insufficient (27.5 — 50 nmoIJL) circulating concentrations of 250HD, respectively, during late pregnancy. A lower maternal 250HD concentration was associated with reduced whole-body and lumbar-spine bone mineral content in children at 9 y. Predictors of maternal 250HD concentration and childhood bone mass were estimated UVB exposure and maternal use of vitamin D supplements. Reduced childhood bone mass was also predicted by reduced concentration of umbilical-venous calcium. The authors concluded that children born to vitamin D deficient mothers were more likely to have low bone mineral content which may increase their subsequent risk of osteoporosis (25). Although vitamin D deficiency increases neonatal hypocalcemia, it is unclear 34 whether there is a cause and effect relation between vitamin D insufficiency and hypocalcemia (168, 186, 187). In a study conducted in Chennai, India, 13 cases of symptomatic hypocalcaemia (n = 50) were present in exclusively breastfed infants and most of them with seizures. None of the infants had received vitamin D supplementation and all their mothers had vitamin D deficiency (250HD < 25 nmoVL) (188). Studies conducted in Turkey and UK also showed similar results (189). Additionally, vitamin D deficiency leads to secondary hyperparathyroidism in the mothers and tetany in the newborns (190). 2.7.4.1 Rickets Vitamin D deficiency leads to rickets in infants and children. Rickets is the most serious clinical consequence of vitamin D deficiency. It is characterized by soft and weakened bones, resulting from poor mineralization of newly fonned bone tissue (49). An early indication of rickets in infants less than 12 months of age is the late closure of the fontanelle (191). Later, rickets is characterized by growth retardation, widening at the end of long bones, and skeletal deformations including the rib cage, spine, anns and legs causing the characteristic bowed legs or knocked knees (192). Most cases of rickets have serum 250HD concentrations less than 10 nmob’L, and elevated alkaline phophatase (a marker of bone formation) and parathyroid hormone concentrations (192, 193). Rickets also leads to hyperparathyroidism and alterations in calcium and phosphorus absorption and metabolism. If rickets is diagnosed at an early stage, effective treatment can be implemented. Untreated rickets may result in permanent skeletal deformities, such as deformity of the pelvis (194). Rickets is uncommon in newborns and it occurs mostly in young children 6-24 months (194). The prevalence of rickets varies considerably in different countries. 35 Rickets is recognized as a common disease of childhood in some parts of Asia. For example, in Northern China during the 1970s, there was a 50% prevalence of rickets in children under three years (195). In a number of developed countries, rickets was once thought to be almost eradicated (196); however, it is present again in neonates and young children in many parts of the world such as Europe and the USA. This is probably due to an increase in dark-skinned immigrants to countries of high latitude (125, 197), breastfeeding for more than 6 months without vitamin D supplementation (11, 196, 198), and avoidance of direct sunlight (199). In a cohort study in 2002-2004, a total of 2325 Canadian pediatricians were surveyed monthly for two years through the Canadian Pediatric Surveillance Program to determine the incidence of rickets. There were 104 confirmed cases of vitamin D-deficient rickets. The overall annual incidence rate was 2.9 cases per 100,000. The authors claimed that this incidence rate may be underestimated because the response rate was 85%, and in addition the survey was sent only to pediatricians and pediatric subspecialists, but not to family doctors. The incidence rates were highest among children residing in the north (Yukon Territory, Northwest Territories, and Nunavut). Intermediate or darker skinned children and breastfed infants without vitamin D supplementation also had higher incidence rates of rickets (24). Maternal vitamin D status during pregnancy and lactation is very important with regard to rickets prevention. Rickets may occur when there is vitamin D deficiency as a result of insufficient transfer of 250}{D from the mother to the fetus, which mostly occurs during the third trimester. Since breast milk only contains about 0.63 j.tg/L or less of vitamin D, infants born to mothers with limited sun exposure and suboptimal vitamin D status as well as breastfed infants without vitamin D supplementation are at higher risks of developing rickets (24). 36 2.7.5 Non-skeletal health of infants Vitamin D deficiency influences non-skeletal health of infants. Poor vitamin D status of infants has been associated with T1D, multiple sclerosis, infant birth weight, and mental disorders. Other conditions in infancy, such as slowing of neonatal cardiac development (200) and asthma (201, 202), are also related to vitamin D deficiency, but the following review will focus on the major complications mentioned above. 2.7.5.1 Type 1 diabetes T1D or insulin dependent diabetes mellitus, is associated with autoimmune destruction of the insulin producing f3 cells in the pancreatic islets (203). Although human fetuses might not suffer from skeletal problems from vitamin D deficiency and insufficiency, they could have an increased risk of non-skeletal problems, such as Ti D, in childhood (168). Vitamin D deficiency and Ti D share some similar risk factors, for example, both are more prevalent in higher latitudes and during spring and winter. At the cellular level, 1,25(OH)D, is involved in the regulation of the endocrine pancreas not only via the plasma calcium levels but also through direct action on the beta cell (204). It is also suggested that 1 ,25(OFI)Dinduces ‘protective’ T helper 2 cell population against the progression of T1D and suppresses T helper 1 cytokine production, thereby preventing -ce1l destruction (204). In a study among north Indian children, 50 children with T1D were matched with 50 healthy controls. The mean 250HD concentration was significantly lower in patients as compared to their controls (50.1 ± 26.6 vs. 65.4 ± 30.7 nmolfL; P 0.009). Approximately 58% of patients had 250HD concentrations below 50 nmol!L and only 32% of controls were below this concentraiton (205). Vitamin D intake during pregnancy is found to be negatively related to the risk of T1D as an increase in vitamin D intake 37 reduces the risk of developing islet autoantibodies in the offspring (179). In a case control study of 85 subjects with T1D and 1071 controls, the risk of T1D was lower in children born to women who had taken cod liver oil during pregnancy (odds ratio 0.36, 95% CI 0.14-0.90) (206). However, long chain n-3 fatty acids in cod liver oil may have some anti-inflammatory properties as well. It was suggested in one article that children supplemented with vitamin D in their first year of life had a lower risk of developing T1D compared with children who were not (207). The dose of vitamin D supplementation matters and an amount of 10 ig/d does not show a protective effect, and therefore, higher doses are being suggested (162, 208). In a large prospective study, children (n = 10366) born in Northern Finland in 1966 were provided with 50 jig of vitamin D per day in the first year of life, and then they were followed for 31 years. Vitamin D supplementation resulted in an 80% reduction in the development of T1D (relative risk 0.16; 95% CI 0.03, 0.51). However, the number of umsupplemented children was very small (n = 32) and only two of those developed T1D (207). In a meta-analysis, the authors concluded that the risk of developing T1D was reduced by 29% in subjects who were supplemented with vitamin D. However, there are a number of weaknesses of the studies included in the meta-analysis, such as retrospective design, no objective measure of 250HD concentration, and use of healthy controls without prior checking (5). 2.7.5.2 Multiple sclerosis Multiple sclerosis (MS) is an inflammatory autoimmune disorder of the central nervous system (209). The incidence of MS increases with increasing latitudes and it is also associated with seasonal variations (210, 211). The authors of the US Nurses Health Study suggest that sunlight exposure and resulting increase in vitamin D may reduce the risk of MS (212). Others have hypothesized that vitamin D reduces the risk of MS by 38 strengthening the immune system against viral infections, a theoretical etiological factor in MS (213-215). Vitamin D deficiency during pregnancy and early years may increase a child’s risk of developing MS in later life. In a population based study, which included Canadian (n = 17874) and British patients (n = 11502) with MS, significantly fewer (8.5%) people with MS were born in November and more (9.1%) were born in May (211). Similarly, a recent cohort study in Sweden has found that more cases with MS were born in June than December and January. The results may reflect seasonal deficiency in maternal concentrations of 250HD which would be expected to be lower in June following the winter than in December and January (216). However, 250HD concentration was not measured in these studies and more research is required to determine whether there is a causal relationship between maternal 250HD and subsequent risk of MS in the offspring. 2.7.5.3 Infant birth weight Some epidemiological studies have demonstrated an association between maternal vitamin D status and infant birth weight. Heavier infants are more likely to be born in the spring than other seasons of the year (217-219). A seasonal effect on maternal vitamin D concentration has been proposed as an explanation for this association. Vitamin D inhibits cellular proliferation and promotes differentiation (48); therefore, inadequate vitamin D during pregnancy may increase cell number leading to a heavier infant. Several animal studies have shown that the offsprings of rats (220) and guinea pigs (221) fed a vitamin D deficient diet during pregnancy had increased birth weight compared to the control animals. Similarly, vitamin D deficient infants (250HD < 37.5 nmol/L) were heavier than vitamin D adequate infants (3698 vs. 3399g; P 0.022) (222). In contrast, vitamin D intake among pregnant women living in Calgary, Canada 39 (n = 279) was positively related to birth weight of newborns. The birth weight of the infants born to women with low milk consumption (< 250 mL/d), regarded as those least likely to consume a daily vitamin D intake of 5 jig, was lower when compared with those born to women who consumed more milk (13.1 ± 4.5 jigld of vitamin D). The study also found that each additional cup of milk was associated with a 41 g increase in birth weight and each additional microgram of vitamin D was associated with an 11 g increase in birth weight (223). In a group of European and Polynesian pregnant women from northern New Zealand (n = 439), infant birth weight was positively related with increasing vitamin D intakes in month 4 of their pregnancy (224). In a more recent study, higher maternal vitamin D intake from diet and supplements was associated with increased birth weight among low income and minority pregnant women (n 2251) living in Camden, US. In average, the birth weight of the infants born to women who consumed less vitamin D (<5 jig) was 60 g lower than those born to women who consumed more (155). However, 250HD was not assessed in these studies. Small-for-gestational age (SGA) birth is defmed as live-born infants whose birth weight lies below 10th percentile for that gender and gestational age. Most recently in a nested case-control study of pregnant women with singleton pregnancies who delivered SGA infants (77 white and 34 black) or non-SGA infants (196 white and 105 black), maternal vitamin D deficiency (< 37.5 nmol/L) and sufficiency (> 75 nmol/L) were both associated with increased risk of SGA. The lowest risk was at senun 250H1) concentrations from 60-80 nmoIJL; however, such relation was only found in white, but not black mothers (225). According to the above studies, the relationship between vitamin D and infant birth weight is not clear and more research is needed. 2.7.5.4 Mental disorders Maternal vitamin D deficiency may have long tenn effects on the offspring’s 40 brain function. The brain is able to synthesize its own active form of vitamin D and it also expresses vitamin D receptor (226). Epidemiological studies have shown an increased risk of schizophrenia in populations living at higher latitudes and individuals born in winter or spring, areas and times with less sun exposure and cutaneous synthesis of vitamin D (227). A small but significant risk of schizophrenia in offspring born in winter/spring was described in the Northern Hemisphere (pooled odds ratio 1.07; 95% CI 1.05 — 1.08), but was not confirmed in a meta-analysis of southern hemisphere studies (228, 229). In the Finnish birth cohort study (n = 9114), the use of either regular or irregular vitamin D supplements during the first year of life was associated with a reduced risk of schizophrenia in males (but not females) compared with no supplementation. In males, the use of at least 50 ig of vitamin D was related to a reduced risk of schizophrenia compared to those on lower doses (230). 2.8 Vitamin D Supplementation during Pregnancy and Lactation A number of studies from 1980 to 2009 have examined the effect of vitamin D supplementation during pregnancy and lactation on maternal 250HD concentrations and neonatal outcomes (Table 2.4 & 2.5). 2.8.1 Impact of vitamin D supplementation during pregnancy on maternal 25-hydroxyvitamin D concentrations and neonatal outcomes Several studies have examined the impact of vitamin D supplementation during pregnancy on maternal 250HD concentrations and neonatal outcomes (Table 2.4). In a group of French women (n = 30), the mean 250HD concentration increased from 25 to 65 nmoIfL after they were supplemented with 25 ig/d of vitamin D3 (186). Datta et al supplemented vitamin D-deficient mothers (n = 160) with 20-40 j.tg/d of vitamin D 41 throughout their pregnancy. After supplementation, mean 2501{D concentrations increased from 14.5 to 28 nmolfL at term. The result indicates that the mothers who were deficient in vitamin D at the beginning were still deficient at the end of their pregnancy, even after supplementation with 20-40 jtgld of vitamin D (114). In a group of Asian immigrant mothers (n = 113), 250HD concentrations in subjects receiving 25 jig/d of vitamin D2 were substantially higher than those receiving the placebo (168 and 16 nmol, respectively) at term (231). These studies suggest that a vitamin D supplement> 25 jtg/d may be required to replete vitamin D status of deficient mothers. Among other studies summarized in Table 2.4, supplement doses ranged from 10 to 30 tg/d. Since initial and/or follow up 250HD concentrations were not determined in these studies, it was difficult to compare their results to the studies mentioned above. One of the confounding factors in these studies is the type of vitamin D supplement used as vitamin D3 is more effective at raising serum 250HD concentrations than vitamin D2 (40). In addition, compliance may have been an issue in these studies. Marya et al (232) studied 120 Indian pregnant women in their 3’ trimester. These women were given either vitamin D supplement (30 jtg/d or two bolus doses of 15000 jig) or placebo. There were significant decreases in maternal alkaline phosphatase and cord blood alkaline phosphatase concentrations among subjects who received the supplementation. Mean birth weights were higher in the infants whose mothers received vitamin D supplementation compared with the placebo group. Another study involving 200 Indian women, also conducted by Marya et al (233), found similar results. It was found that infants whose mothers received supplementation (two bolus doses of 15000 jig) had greater intrauterine growth, with greater crown-heel length, head and arm circumference, and skin-fold thickness. However, 250HD concentrations were not determined in these studies. In a group of Scottish pregnant women (n = 1139), neonatal 42 hypocalcemia was more common in the placebo group compared with subjects receiving 10 ig/d of vitamin D2 (234). Brooke et al (231) reported similar results with regard to neonatal hypocalcemia. The infants of the mothers who received the placebo had larger fontanelles, suggesting impaired ossification of the skull, when compared with those born to mothers supplemented with 25 jigld of vitamin 1)2. The mean maternal plasma calcium concentration was higher in the supplemented group at delivery compared with the placebo group. Yu et al (235) supplemented Indian Asian, Middle Eastern, black, and Caucasian mothers (n = 180) with vitamin D (20 jig/d D2 or 5000 jig/once D3) or placebo. The mean cord blood 250HD concentration was higher in groups receiving supplement. At delivery, there were less cases of secondary hyperparathyroidism among women who were supplemented with vitamin 1). 2.8.2 Vitamin D supplementation during lactation Vitamin 1) content of human breast milk has been found to be quite low; therefore, breast milk is insufficient to provide adequate vitamin 1) for the newborn for the duration of exclusive breastfeeding. Health Canada recommends that breastfed infants are supplemented with 10 j.tg/d of vitamin D (236). However, according to the NHANES 1999-2002, less than 10% of American mothers supplemented their babies with vitamin D (237). Further, anecdotal evidence suggests that some mothers and even healthcare professionals are reluctant to supplement breastfed infants with vitamin D as it implies that breast milk is less than a complete food for infants. It appears that vitamin D supplementation to mothers during lactation improves vitamin D content in human milk, which may protect the infant against low 250H1) during early infancy (238-240). In a study in Finland, 49 pairs of mother and infant were randomized into three groups. Mothers were supplemented with either 25 or 50 jig/d of 43 vitamin D3 or infants were given 10 jigld of D3 for 15 weeks. Serum 250HD increased in nursing infants of all three groups, and was greater in the women receiving 50 ,.tg/d than those receiving 25 tg!d. In the third treatment group, only infants were supplemented with 10 jigld; however; the increase in the mean 250111) concentration in these infants was the greatest indicating that supplementing the infants directly was more effective in raising their 250111) concentrations (147). These results were consistent with Rothberg et al (241) and Saadi et al (242). In a recent study in the US, mothers were given either 40 tg!d D2 + 10 jig/d D3 or 90 jig/d D2 + 10 jig/d D3 for 3 months. Maternal serum 250HD concentrations increased in both groups with a greater increase in the group receiving 100 j.tg/d of vitamin D. Infant serum 250HD also increased in a dose responsive manner (243). As summarized in Table 2.5, the vitamin D supplement doses ranged from 10 ig/d to as high as 160 g/d. These studies showed that supplementing lactating women with vitamin D improved maternal and infant vitamin D content and directly supplementing infants may be a more effective way to raise their 250HD concentrations than supplementing their mothers. In summary, obtaining adequate vitamin D during pregnancy is very important for health of both mother and child. Low vitamin D status during pregnancy, based on low circulating concentrations of 250HD, has been reported in several countries. However; there were few studies of pregnant women in Canada; yet there is reason to suspect a high rate of vitamin D insufficiency. Given the importance of vitamin D during pregnancy and the lack of studies assessing the vitamin D status of Canadian women the primary objective of this research is to determine the prevalence of vitamin D deficiency and insufficiency in a group of Vancouver pregnant women. Factors affecting vitamin D status such as season, ethnicity, and vitamin D intake will be examined. The few studies that examined the relationship between PTH and 250HD have found inconsistent results. 44 Additionally, there are limited studies that measured skin color directly. Therefore, the relationship between 250HD and PTH as well as skin colour will also be examined. It is hoped that the results of the proposed study will provide more information in the areas mentioned above. 45 Table 2.1 Food sources of vitamin D (150) Food Serving Vitamin D (,ig) Fortified Milk 1 cup 2.5 Fortified orange juice 1 cup 2.3 Fortified rice or soy beverage 1 cup 2.0 Fortified margarine 2 tsp 1.3 Egg yolk 1 each 0.6 Tuna, bluefin, cooked 75 g 17.3 Salmon, canned or cooked* 75 g 15.2 Tuna, skipjack, cooked 75 g 9.5 Sardines, Pacific, canned 75 g 9.0 Salmon, Atlantic, cooked 75 g 5.6 Herring or trout, cooked 75 g 3.9 Tuna, canned, yellowfin (albacore, 75 2 6 ahi) g Mackerel, cooked 75 g 2.0 Sardines, Atlantic, canned 75 g 1.8 Tuna, canned, light or white 75 g 1.0 * includes Chinook, Coho, Humpback (pink), Sockeye 46 Ta bl e 2. 2 V ita m in D st at us o fp re gn an ta n d la ct at in g w o m en R ef er en ce Ci ty ,c o u n tr y & la tit ud e (y ea r) G ro up Se ru m 25 -h yd ro xy vi ta m in D (nm olJ L) W ai te rs et a! (10 6) In uv ik zo n e, Ca na da , 6 8° N Pr eg na nt w o m en ; 1- 8 m o n th ge st at io n M ea n ± SD In ui ts (n 51 ) 48 .8 ± 14 .2 N at iv e In di an s (n = 37 ) 52 .1 ± 25 .9 Ca uc as ia ns (n 33 ) 59 .8 ± 29 .4 Le br un et al (10 7) M an ito ba ,C an ad a, 49 °N N at iv e la ct at in g w o m en (n = 80 ) M ea n 19 .8 G en ui s e ta l( 10 8) Ed m on to n, Ca na da , 53 °N (Ju ne Pr eg na nt w o m en (n = 83 ) 23 % < 40 20 01 — M ar ch 20 07 ) 53 % 40 — 80 24 % 80 — 25 0 Sl ok a et al (11 0) N ew fo un dl an d & La br ad or , Ca uc as ia n pr eg na nt w o m en M ea n Ca na da ,4 7° N & 52 °N W in te r( Jan ua ry — M ar ch 20 07 , n = 30 4) 52 .1 Su m m er (Ju ly — Se pt em be r2 00 7, n = 68 .6 28 9) N ew ho ok et al (10 9) N ew fo un dl an d & La br ad or , Ca uc as ia n pr eg na nt w o m en M ea n ± SD Ca na da ,4 7° N & 52 °N Su m m er (S ep tem be r2 00 5, n = 25 ) 61 .1 ± 17 .8 W in te r(M ar ch 20 06 ,n =2 5) 51 .9 ± 17 .9 Bo dn ar et al (26 ) Pi tts bu rg h, U S, 40 °N (19 97 — W hi te pr eg na nt w o m en (n = 20 0) M ea n (ra ng e) 20 01 ) A tte nn 80 .4 (7 6.0 — 85 .1) 4- 21 w ee k ge st at io n 73 .1 (69 .4 — 78 .9 ) B la ck pr eg na nt w o m en (n = 20 0) A t t er m 49 .4 (46 .1 — 52 .9 ) 4- 21 w ee k ge st at io n 40 .2 (37 .9 — 42 .7 ) R ef er en ce Ci ty ,c o u n tr y & la tit ud e (y ea r) G ro up Se ru m 25 -h yd ro xy vi ta m in D (n mo l/L ) D av is et a! (11 1) B al tim or e, M ar yl an d, U S, 39 °N Pr eg na nt A fri ca n A m er ic an ad ol es ce nt s M ea n ± SD (a e 16 .5 ± 1.1 y) 2 tr im es te r( 18 ± 1.8 w ee k, n = 44 ) 52 .4 ± 20 .5 3rd tr im es te r( 28 .4 ± 2.1 w ee k, n = 36 ) 56 .3 ± 19 .8 G al e et al (11 3) So ut ha m pt on , U K , 50 °N (19 91 Ca uc as ia n pr eg na nt w o m en M ed ia n (qu art ile s) — 19 92 ) 28 — 42 w ee k ge st at io n (n = 46 6) 50 (30 ,7 5. 3) D at ta et al (11 4) Ca rd iff , So ut h W al es , 51 ° N A fri ca n, A fro -C ar ib be an ,F ar -E as te rn , M ea n ± SD (A pr 19 95 — A pr 19 96 ) M id dl e- Ea ste rn ,I nd ia n su bc on tin en t pr eg na nt w o m en A tf irs t a n te na ta lv isi t( n = 16 0) 14 .5 ± 1 O ’R io rd an et al (11 5) Co rk , Ir el an d, 52 °N (20 04 — Pr eg na nt Ca uc as ia ns (n 43 ) M ea n ± SD 20 06 ) 1st tr im es te r 39 .2 ± 17 .9 2K tr im es te r 44 ± 22 3rd tr im es te r 53 .3 ± 28 .4 M ad el en at et al (11 6) Fr an ce ,4 6° N (w int er o f 1 99 9) Pr eg na nt w o m en ; 27 -3 2 w ee k ge sta tio n M ea n ± SD (n =5 9) 35 .5 ±2 1 Ch al la et al (11 7) G re ec e, 40 °N M ea n ± SD La ct at in g w o m en Ju ne — N ov (n = 35 ) 32 .2 ± 3.3 La ct at in g w o m en D ec — M ay (n 31 ) 27 ± 2. 5 N ic ol ai do u et al A th en s, G re ec e, 38 °N (Ju ne M ed ia n (qu art ile s) (11 8) 20 03 — M ay 20 04 ) Pr eg na nt w o m en ; at de liv er y (n = 12 3) 41 (27 .5, 52 .8 ) D el iv er in su m m er /fa ll 47 .3 (32 .3, 46 .3 ) D el iv er in w in te r/s pr in g 36 .5 (25 .3, 46 .3 ) 00 A ar hu s, D en m ar k, 56 °N (sp rin g an d su m m er o f 2 00 3) La ct at in g Ca uc as ia n w o m en (n = 89 ) Sp rin g La te su m m er W in te r M ed ia n (qu art ile s) 80 .5 (53 .5, 11 1) 90 .5 (70 .8, 11 6. 3) 58 .5 (39 .5, 74 ) H en rik se n et al (12 0) O slo , N or w ay , 59 °N (O ct 19 91 — Ja n 19 92 ) 18 w ee k ge st at io n Pr eg na nt Pa ki sta ni im m ig ra nt s (n = 38 ) N or w eg ia n pr eg na nt w o m en (n = 38 ) M ed ia n 19 55 M ad er et al (12 1) O slo & D ra m m en , N or w ay , 59 °N (M ar 20 04 — Fe b 20 06 ) La ct at in g im m ig ra nt w o m en Pa ki sta ni (n = 45 ) T ur ki sh (n 25 ) So m al i ( n= 10 ) M ea n ± SD 26 .7 ± 16 .5 26 .1 ± 14 .1 21 .5 ± 12 .1 V an de r M ee r et al (12 2) Th e H ag ue , N et he rla nd s, 52 °N (Ju n 2 00 2 — M ar 20 04 ) Pr eg na nt w o m en (n 35 8) 12 w ee k o f g es ta tio n W es te rn Tu rk ish M or oc ca n O th er n o n -w es te rn M ea n± SD 52 .7 ± 21 .6 15 .2 ± 12 .1 20 .1 ± 13 .5 26 .3 ± 25 .9 W ie ld er s et al (12 3) A m er sf oo rt, N et he rla nd s, 52 °N (A pr 20 04 — A pr 20 05 ) Pr eg na nt w o m en ; 10 an dl or 30 ge st at io n D ut ch /E ur op ea n o rig in (n 54 5) N on -w es te rn o rig in (n = 13 1) w ee k 1, 25 di hy dr ox yv ita m in D (nm oL fL ) 5% < 20 55 % < 20 Ca va lie r e t a ! ( 12 4) Li eg e, B el gi um , 50 °N (N ov 20 06 ) R ec en tly pr eg na nt w o m en (n = 24 ) La ct at in g m o th er s (n = 65 ) 25 -h yd ro xy vi ta m in D (nm oIJ L) 88 % < 30 86 % < 30 M øl le r e t a l ( 11 9) R ef er en ce Ci ty , c o u n tr y & la tit ud e (y ea r) G ro up Se ru m 25 -h yd ro xy vi ta m in D (n mo lIL ) R ef er en ce Ci ty , c o u n tr y & la tit ud e (y ea r) G ro up Se ru m 25 -h yd ro xy vi ta m in D (n mo l/L ) G ro ve r & M or le y (27 ) M el bo ur ne , A us tra lia , 37 °S (Ju l 19 99 — A pr 20 00 ) V ei le d an ch or da rk -s ki nn ed pr eg na nt w o m en (n = 82 ) M ed ia n (qu art ile s) 14 (3, 77 ) M or le y et al (33 ) M el bo ur ne , A us tra lia , (A pr 20 02 — Se p 20 03 ) 37 °S 11 w ee k ge st at io n (n =3 59 ) 28 -3 2 w k o fp re gn an cy (n = 37 4) & R od da M el bo ur ne ,A us tra lia , 3 7° S (Ju n 19 94 — Fe b 19 99 ) A fri ca n, In di an /P ak ist an i, Ea st er n, Ita lia n, Eu ro pe an pr eg na nt w o m en (n = 31 ) M id dl e 8 l% 2 5 de sc en t 90 % 40 Ju dk in s & Ea gl et on (12 6) W el lin gt on , N ew Ze al an d, 41 ° S (20 05 — 20 06 ) A fri ca n, M ao ri, Eu ro pe an , M id dl e Ea st er n, Po ly ne si an pr eg na nt w o m en (n = 90 ) 87 % < 50 61 .2 % < 25 B ei jin g, Ch in a, 40 °N (su mm er o f 1 98 3) La ct at in g m o th er s (n = 54 ) Sa ch an et al (12 8) Lu ck no w ,N or th er n In di a, 26 °N (S ep — N ov 20 02 ) U rb an & ru ra l pr eg na nt w o m en ; at de liv er y (n = 20 7) M ea n± SD 35 ± 23 .3 A tiq et al (12 9) K ar ac hi ,I nd ia ,2 4° N (19 93 ) La ct at in g w o m en (n 62 ) U pp er so ci o- ec on om ic (n = 37 ) Lo w er so ci o- ec on om ic (n = 25 ) M ea n± SD 26 .5 ± 24 .1 39 .8 ± 15 .7 Se th e ta l(1 30 ) B as si re ta l( 13 1) N ew D el hi ,I nd ia ,2 8° N Te hr an , Ira n, 35 °N (Ja n — Se p 19 97 ) La ct at in g w o m en (n = 18 0) Pr eg na nt w o m en ; at de liv er y (n = 50 ) M ea n ± SD 27 .2 ± 14 .6 M ea n± SD 12 .8 ± 26 N oz za (12 5) 6. 4% < 28 7. 2% < 28 H o et al (12 7) M ea n 50 -6 0 L J R ef er en ce K az em i e ta ! ( 13 2) Ci ty , c o u n tr y & la tit ud e (y ea r) Te br an , Ira n, 35 °N (w int er & su m m er ) Se ru m 25 -h yd ro xy vi ta m in D (nm olJ L) M ea n± SD 19 .4 ± 3. 9 M ag hb oo li (13 7) et a! Te br an , Ira n, 35 °N (w int er o f 20 02 Pr eg na nt w o m en ; at de liv er y (n 55 2) M ea n ± SD 27 .8 ± 21 .7 Pe hl iv an et al (13 3) K oc ae li, Tu rk ey ,4 1° N Pr eg na nt w o m en ; la st se m es te r (n = 78 ) M ea n± SD 17 .5 ± 10 .3 M uk am el et al (13 4) Te l A vi v & Bn ei B ra k, Is ra el , 32 °N (la te su m m er of . 19 98 — th e sp rin g o f 1 99 9) Je w ith Is ra el i m o th er s O rth od ox (n = 15 6) N on -o rth od ox (n = 18 5) M ea n± SD 33 .8 ± 18 .8 46 .5 ± 24 D aw od u et al (13 5) A l Am , U ni te d A ra b Em ira te s, 24 °N (A pr — O ct 19 99 ) A ra b/ So ut h A sia n la ct at in g w o m en (n = 90 ) M ed ia n (qu art ile s) 21 .8 (14 .8, 34 ) Ta ha et a! (13 6) Ri ya dh , Sa ud i A ra bi a, 24 °N (P re 19 84 ) La ct at in g w o m en (n = 10 0) M ea n ± SD 33 .3 ± 35 .8 Se re ni us et al (94 ) Ri ya dh , Sa ud i A ra bi a, 24 °N (P re 19 84 ) U pp er cl as s la ct at in g w o m en (n = 27 ) M id dl e cl as s la ct at in g w o m en (n = 58 ) Lo w er cl as s la ct at in g w o m en (n = 33 ) M ea n (ra ng e) 17 .5 (8 — 50 ) 13 .8 (7. 5 — 54 .5 ) 12 (7. 5— 50 .5 ) Sa nc he z e ta ! (13 8) M ai du gu ri, N ig er ia , 11 ° N (Ju ly 19 94 ) Pr eg na nt te en ag er s; at de liv er y 1s t t rim es te r( n = 10 ) 2n d tr im es te r ( n 10 ) 3r d tr im es te r ( n = 10 ) H ea lth y n o n pr eg na nt w o m en (n 21 ) M ea n± SD 24 .2 ± 8.1 43 ± 19 .8 74 .5 ± 26 .3 40 .3 ± 9.5 G ro up Pr eg na nt w o m en ; at de liv er y (n = 67 ) Table 2.3 Variation in recommended vitamin D intake for (non-high-risk) pregnant and lactating women Europe [European Union (1992)] 42) UK [COMA (1996)] (143) UK [UK Dept of Health (2008)] (144) Australia and New Zealand [NHMRC (2005)] (145) US, United States; FNB, Food and Nutrition Board; UK, United Kingdom; COMA, Committee on the Medical Aspects of Food Policy; NHMRC, The National Health and Medical Research Council Country jReference] US [FNB (1997)] (29) World Health Organization (2002) (141) Canada [Health Canada (2004)] (236) Canada [Canadian Pediatric Society (2007)] (13) Recommend Intake 5 ig/d 5 jig/d 5 jig/d Consider 50 ig/d (especially in winter) 10 ig/d 10 ig/d 10 jig/d 5 pg/d 52 Ta bl e 2. 4 Su m m ar y o f v ita m in D su pp le m en ta tio n st ud ie sd ur in g pr eg na nc y R ef er en ce L oc at io n Po pu la tio n (n) St ud y Ty pe V ita m in D Su pp le m en t 25 0H ED (n mo l/L ) O th er o u tc om es & D os e D ur at io n Fo rm In iti al E nd po in t* L at itu de (n) M ar ya et In di a, H in du w o m en R T 30 tg /d 3r d D2 N D N D B irt hw ei gh t in bo th D v s. al (23 2) 29 °N (12 0) (25 ) tr im es te r pl ac eb o; A lk al in e 15 00 0 1x in N D N D ph os ph at as e . & m at er na l g (20 ) ea ch o f 7 an d co rd c a lc iu m t in D & 8 m o (15 00 0 jig )v s. pl ac eb o Pl ac eb o 3r d N D N D (75 ) tr im es te r M ar ya et In di a, A si an -I nd ia n RT 15 00 0 1 x in D3 N D N D IM at er na l& co rd ca lc iu m al (23 3) 29 °N (30 0) jig ea ch o f 7 an d . al ka lin e ph os ph at as e (20 0) & 8 m o w ith D Pl ac eb o 1x in N D N D B irt hw ei gh t, cr o w n -h ee l (10 0) ea ch o f7 le ng th ,h ea d an d ar m & 8 m o ci rc um fe re nc e w ith D Co ck bu rn Sc ot la nd , Sc ot tis h (11 39 ) QR T 10 jig /d Fr om 12 D2 N D 43 In fa nt 25 0H D at 6t 1 da y et al (23 4) 55 °N (50 6) w k w ith D ;s ym pt om at ic Pl ac eb o Fr om 12 N D 33 hy po ca lc em ia 0. 4% w ith D (63 3) w k v s. 0. 9% w ith pl ac eb o B ro ok e et En gl an d, A sia n (11 3) D B R CT 25 j.tg /d Fr om 28 D2 20 16 8 Co rd bl oo d 25 0H D in D ; al (23 1) 51 ° N (39 ) w k 5 ca se s o fs ym pt om at ic Pl ac eb o Fr om 28 20 16 hy po ca lc em ia in pl ac eb o (55 ) w k gr ou p M al le t e t a! (24 4) In di an A sia n (45 ), M id dl e Ea st er n (45 ), B la ck (45 ) & Ca uc as ia n (45 ) Fr an ce , Fr en ch (16 0) 45 -4 8° N 3r d tr im es te r o n ce 7th m o (1 5) tr im es te r Se co nd ar y hy pe rp ar at hy ro id ism . w ith D ; Co rd 25 0H D I w ith D Co rd an d in fa nt (4 d) 25 0H D tw it hD Yu et al (23 5) En gl an d, 51 ° N R ef er en ce Lo ca tio n Po pu la tio n (n) St ud y Ty pe V ita m in D Su pp le m en t 25 0H D (nm oIJ L) O th er o u tc om es & D os e D ur at io n Fo rm In iti al E nd po in t* L at itu de (n) RT D3 N D 34 50 00 jig (60 ) O nc e in 27 w k 20 j.tg /d (60 ) Fr om 27 w k N o (60 ) Fr om 27 w k RT 25 p.g /d (21 ) 50 00 jig (27 ) N o (29 ) 3rd Fr en ch (-- 30 ) RT D el vi n et a! (18 6) D at ta et al (11 4) 1)2 N D 42 N D 27 D2 N D 9 D2 N D 25 N D 26 D3 — 25 65 - — - 25 33 N D 14 .5 28 Co rd 25 0H D w ith 1) Fr an ce , 45 °N U ni te d K in do m , 52 °N 25 jig ld (‘— 15) N o tr im es te r 3rd tr im es te r 3rd N on -E ur op ea n m in or iti es (80 ) In te rv en tio n Tr ia l 20 — 40 jig /d (80 ) Fr om fir st an te na ta l v isi t _ _ _ _ 25 01 {D , 2 5 hy dr ox yv ita m in D ; RT , ra n do m iz ed tri al; OR T, qu as i ra n do m iz ed tr ia l; D BR CT , do ub le -b lin d ra n do m iz ed co n tr ol tr ia l; N D , n o t de te rm in ed * A ll m ea su re m en ts at te rm . V al ue s re la tiv e to in iti al 25 0H D co n ce n tr at io n w he re in iti al co n ce n tr at io n w as m ea su re d. Pa ra th yr oi d ho rm on e le ve ls u n ch an ge d af te r su pp le m en ta tio n w ith D Ta bl e 2. 5 Su m m ar y o f v ita m in D su pp le m en ta tio n st ud ie s du rin g la ct at io n R ef er en ce Lo ca tio n Po pu la tio n St ud y V ita m in D Su pp le m en t 25 01 1D (nm oIJ L) O th er O ut co m es & (n) Ty pe In fa nt /M ot he D os e & In fa nt /M ot he r (n) D el iv er y Fo llo w L at itu de r D ur at io n u p A la -H ou ha Fi nl an d, M ot he r & R T M ot he rs 25 j.tg /d D3 M ot he rs (15 ) — 50 — 43 N o sig ni fic an t la et al 62 °N In fa nt Pa irs 20 w k In fa nt s (15 ) — - 43 — 30 in te rg ro up di ffe re nc es (14 6) Su m m er : In fa nt s 10 jig /d D3 M ot he rs (16 ) — — 43 — 33 in ca lc iu m o r al ka lin e Ju l-D ec (45 ) 20 w k In fa nt s (16 ) — 38 — 10 0 ph os ph at as e. M ilk In fa nt s 25 ig /d D3 M ot he rs (14 ) — 50 — 38 an tir ac hi tic ac tiv ity o f 20 w k In fa nt s (14 ) — 33 — 13 3 m o th er s w ith 25 jig /d W in te r: M ot he rs 25 j.tg /d D3 M ot he rs (17 ) — 23 — 63 w as in su ff ic ie nt in Ja n- A pr (47 ) 20 w k In fa nt s (17 ) — 25 - — 30 w in te r In fa nt s 10 j.tg /d D3 M ot he rs (15 ) - — 23 — 50 2O w k In fa nt s (15 ) — 20 — 80 In fa nt s 25 . tg /d D3 M ot he rs (15 ) ‘— 23 — 38 : 2O w k In fa nt s (15 ) — 25 — 11 0 A la -H ou ga Fi nl an d, M ot he r & RT M ot he rs 50 jig /d D3 M ot he rs (17 ) - — 28 - — 90 N o sig ni fic an t la et a! 62 °N In fa nt Pa irs 15 w k In fa nt s (17 ) - — 23 — 73 in te rg ro up di ffe re nc es (14 7) W in te r: M ot he rs 25 gg /d D3 M ot he rs (16 ) — 25 — 75 in m at er na l o r in fa nt Ja n- A pr (49 ) 15 w k In fa nt s (16 ) — 18 — 38 ca lc iu m , p ar at hy ro id In fa nt s 10 jig /d D3 M ot he rs (16 ) — 25 — 30 ho rm on e o r al ka lin e 15 w k In fa nt s (16 ) - — 20 — 80 ph os ph at as e G re er et a! U ni te d M ot he r & D B R In fa nt s 10 ig /d In fa nt s (9) — 73 - — 98 Si gn ifi ca nt ly hi gh er (24 5) St at es , In fa nt Pa irs CT bo ne m in er al co n te nt in 39 °N (18 ) th e su pp le m en tg ro up In fa nt s Pl ac eb o In fa nt s (9) — 50 — 50 R ef er en ce Lo ca tio n Po pu la tio n St ud y V ita m in D Su pp le m en t 25 0H D (n mo l/L ) O th er O ut co m es & (n) Ty pe In fa nt /M ot he r D os e & In fa nt /M ot he r (n) D el iv er y Fo llo w L at itu de D ur at io n u p R ot hb er g So ut h M ot he r & D B R M ot he rs Pl ac eb o 6 M ot he rs (10 ) 30 25 In fa nt 25 0H D w as (24 1) A fri ca , In fa nt Pa irs CT w k In fa nt s (10 ) 22 3 u n af fe ct ed by 12 .5 o r 25 26 °S W hi te (77 ) tg /d m at er na l M ot he rs 12 .5 . tg /d M ot he rs (9) 30 35 su pp le m en ta tio n. D3 6 w k In fa nt s (9) 22 26 Su pp le m en tin g in fa nt s M ot he rs 25 tg /d D3 M ot he rs (9) 30 37 w ith 10 jig ld di re ct ly 6 w k In fa nt s (9) 22 24 si gn ifi ca nt ly ra ise d In fa nt s 10 ig /d D3 M ot he rs (17 ) 30 28 in fa nt 25 0H D . 6 w k In fa nt s (17 ) 22 38 H ol lis & U ni te d M ot he r& M ot he rs 40 g /d D2 M ot he rs (9) 69 90 ti n m ilk an tir ac hi tic W ag ne r St at es , In fa nt Pa irs + 10 j.tg /d In fa nt s (9) 20 70 ac tiv ity fr om 35 .5 to (24 3) 32 °N (18 ) D3 m o 69 .7 1U /L M ot he rs 90 p.g /d D2 M ot he rs (9) 82 1 1 1 in m ilk an tir ac hi tic + 10 j.tg /d In fa nt s (9) 34 77 ac tiv ity fr om 40 .4 to D3 m o 13 4. 6 IU /L W ag ne r e t U ni te d M ot he r& RT M ot he rs & 10 ig /d D3 M ot he rs (10 ) 80 * 95 al (24 6) St at es , In fa nt Pa irs In fa nt s 7. 5 j.tg /d D3 In fa nt s (10 ) 33 * 10 8 32 °N (19 ) 6m o M ot he rs 16 0 ig /d M ot he rs (9) 85 * 14 8 D3 In fa nt s (9) 35 * 1 1 5 R ef er en ce Lo ca tio n Po pu la tio n St ud y V ita m in D Su pp le m en t 25 0H D (n mo l/L ) O th er & (n) Ty pe In fa nt /M ot he r D os e & In fa nt /M ot he r (n) D el iv er y Fo llo w O ut co m es L at itu de D ur at io n u p Sa ad ie t a l U ni te d M ot he rs (90 ) RT M ot he rs 50 . tg /d D2 M ot he rs (22 ) N D N D t in m ilk (24 2) A ra b & In fa nt 3m o an tir ac hi tic Em ira te s Pa irs (92 ) ac tiv ity fro m 24 °N u n de te ct ab le M ot he rs & 50 fig /m o D2 M ot he rs (22 ) - 29 4 2 (< 20 IU /L ) In fa nt s 10 jig /d D2 3 In fa nt s (22 ) — 44 - — 50 to 50 .9 lU lL m o (ra ng e 0 — M ot he rs & 15 00 ig /m oD 2 M ot he rs (22 ) - — 22 ‘— 36 62 .5 ) In fa nt s 10 j.tg /d D2 3 In fa nt s (24 ) 14 ‘- — 45 m o Pe hl iv an Tu rk ey , In fa nt s & RT In fa nt s 10 tg /d 14 w k In fa nt s (19 ) N D ‘- — 77 N o et al (13 3) 41 °N Co nt ro ls (65 ) di ffe re nc es in In fa nt s 20 flg /d 14 w k In fa nt s (21 ) N D — 92 le ve ls o f In fa nt s N o In fa nt s (65 ) N D — 42 ca lc iu m , ph os ph or ou s, an d ali ca lin e ph os ph at as e be tw ee n th e su pp le m en te d an d co n tr ol gr ou p 25 0H D ,2 5 hy dr ox yv ita m in D ; R T, ra n do m iz ed tr ia l; OR T, qu as ir an do m iz ed tr ia l; D BR CT ,d ou bl e- bl in d ra n do m iz ed co n tr ol tr ia l; N D ,n o t de te rm in ed * In iti al M ea su re m en ts 2 w k po st -p ar tu m Figure 2.1 The chemical structure of vitamin D2 (ergocalciferol) (247) Figure 2.2 The chemical structure of vitamin D3 (cholecalciferol) (247) 58 SOLAR SOLAR UVB UVB7f 7-DHC . PreD3 Si’Jfl Heat ,. VitaminD3Z... Figure 2.3 Schematic diagram of cutaneous production of vitamin D and its metabolism and regulation for calcium homeostasis and cellular growth (198) DIet chylomicrons Lumisterol Tachysterol 5, 6-transvitamin D Suprasterol 1&2 J25-OHaseJ Maintains Normal Cell Proliferation I)2D 4 ‘ Calcitroic 59 OH Figure 2.4 The chemical structure of calcitriol (1,25 dihydroxyvitamin D) (247) Figure 2.5 The chemical structure of calcidiol (25-hydroxyvitamin D) (247) 60 80 70 60 -i 40 30 20 Mar May Jul Sep Nov Figure 2.6 Serum 25-hydroxyvitamin D concentrations by month, of New Zealand children and adolescents (n 1583) who participated in the 2002 National Children’s Nutrition Survey. Values are means (99% CI) adjusted for age, sex, ethnicity, latitude, and obesity using multiple linear regression, and weighted to account for the complex survey design. *Djfferent from March; P < 0.01 (71). * * * * * * * 61 CHAPTER 3: METHDOLOGY 3.1 Overview of Design This is a cross-sectional study describing the vitamin D status and factors determining 250HD concentrations in a group of BC pregnant women from diverse backgrounds. Participants were asked to complete a demographic and lifestyle questionnaire and a food frequency questionnaire (FFQ) which estimated the intake of vitamin D and calcium in a month. A non-fasting blood sample was collected and skin color was quantified. Anthropometric measures (height and weight) were also collected. 3.2 Sample Size A sample size of 307 would allow the detection of effect sizes as small as 0.05 with up to 8 factors or covariates with 80% power and a two-sided alpha of 0.05 (248). The primary outcome measure was 250HD concentration. We also wanted to ensure we had at least 50 women per season and at least 50% Non-European to determine the effects of season and ethnicity (including skin color), respectively, on 250HD. 3.3 Recruitment and Participant Selection The current research was approved by The University of British Columbia Clinical Research Ethics Board, Vancouver Coastal Health, and Children’s and Women’s Research Review Committee (Appendix A & B) and all women gave informed written consent to participate (Appendix C). A convenience sample of 340 pregnant women were recruited from Vancouver and the Lower Mainland (49°N) between February 2009 and February 2010 through: The BC Women’s Hospital and Health Center; The Healthiest Babies 62 Possible Program; Youth Pregnancy & Parenting Program (Evergreen Community Health Centre); Douglas College prenatal programs (Richmond and Bumaby sites); South Community Health Centre; Three Bridges Community Health Centre; Richmond Health Department; Pacific Spirit Community Health Centre; and other local community programs. These programs provide services to women of a variety of ages, ethnic (i.e., Indigenous, White, Chinese, Iranian, South-East Asian, and the Indian sub-continent) and socio-economic background. Recruitment was through active recruiting at the BC Women’s Hospital (the investigator approached potential participants in waiting areas), brochures placed at clinics, advertisement (newspapers and websites) (Appendix D & E), word of mouth, and through public health dietitians and nurses who served these programs. Pregnant women in between 20th and 35th week of gestation and those with singleton pregnancies and identified for low-risk delivery were eligible to participate in the study. Women were not eligible if they had: any co-morbid conditions such as gestational diabetes, cardiac or renal disease, HIV/AIDS, chronic hypertension, or autoimmune disease; conditions associated with vitamin D malabsorption such as celiac disease. In order to assess the seasonal effect on 250HD concentrations, a similar number of women were recruited in winter, spring, summer, and fall months. Given the diversity of prenatal programs and women served a number of approaches were used to conduct the clinics. Women attended a clinic held concurrently with one of their prenatal classes (i.e. Healthiest Babies Possible Program) or women were assessed individually at the BC Women’s Hospital. A standard protocol was used for each of the participants. All questionnaires were translated into Chinese and a Mandarin-speaker (the investigator) provided language support if required. 63 3.4 Procedures The study involved one clinic visit that took approximately 3 0-60 minutes. An identification number from I to 340 was assigned to each subject. After providing consent participants were asked to complete a demographic and lifestyle questionnaire that included questions on sun exposure (clothing practice, outdoor activity, sunscreen use, and tanning practice), pre-pregnancy weight, date of birth, week of pregnancy (based on ultrasound), estimated pregnancy due date, smoking status, ethnic background, annual income, and education attainment (Appendix F). In cases where the participants indicated that they belonged to more than one ethnic group, a single ethnic category was assigned using a priority system. If a non-European ethnicity was one of the groups reported, the participant was assigned to the non-European ethnic category. If two or more non-European ethnicities were reported, the participant was assigned to the non-European ethnic category that matched with the cultural origin of her ancestors. However, the latter case was uncommon. Heights were measured to the nearest 0.1 cm at the time of interview, using a stadiometer. Body weight was measured to the nearest 0.1 kg in light indoor clothing and no shoes using a floor standing scale. 3.4.1 Assessment of dietary intake Intake of vitamin D and calcium from food sources including fortified foods and supplements in a typical month during pregnancy were estimated using a validated semi-quantitative FFQ (Appendix G). The FFQ was developed and evaluated for rapid assessment of vitamin D intake in a group of Canadian healthy young adults of diverse ancestry during the late winter of 2007 (n 107). Estimated vitamin D intakes from the FFQ 64 correlated well with vitamin D intake from 7-day food records: 6.5 ± 4.4 and 4.3 ± 3.6 jig/d, respectively (r = 0.529, P < 0.001). Vitamin D intakes from the FFQ were also positively related to serum 250HD concentrations (r = 0.48 1, P <0.001) (249). In the FFQ, frequency of consumption was listed as: never or less than 1 per month, 1 per month, 2-3 per month, 1 per week, 2 per week, 3-4 per week, 5-6 per week, 1 per day, and 2+ per day. Serving sizes were small, medium, and large. Participants were asked to provide information on nutritional supplements used in the previous month using as much detail as they could remember. To ensure an accurate assessment of supplement use, women were asked to bring any supplements they were using to the clinic. Brand name of supplement, vitamin D and calcium content, and amount taken was recorded. FFQs were scanned and analyzed by the University of Saskatchewan College of Pharmacy and Nutrition, which developed the original questionnaire. Vitamin D and calcium contents for each food item in the FFQ were determined using ESHA Food Processor (Version 8.0, ESHA Research, Ore), which included the 1997 Canadian Nutrient File from Health Canada. Fortification amounts for foods recently approved in Canada were updated (orange juice, soy beverage) to present values (249). Dietary intake was exported to Excel spreadsheet reports. Vitamin D and calcium content of the supplements were obtained from label declarations. 3.4.2 Skin color measurement An objective estimate of natural (constitutive) and sun-induced (facultative) skin color was measured by reflectance colorimetry using a spectrophotometer (Konica Minolta Sensing Americas, Inc. CM-600d; Made in Japan), which is a handheld tristimulus 65 reflectance colorimeter. This instrument measures skin reflectance of light at 256 points over the visible light wavelength spectrum from 400 to 700 nm, and reports data using the Commission International de l’Eclairage (CIE) Lab system in which colors are described by their lightness value (L*), the amount of green or red (a*), and the amount of yellow or blue (b*) they contain. In practice, the L* value represents relative brightness of color (ranging from black to white) and along with the a* value best captures skin redness; the b* value is used to measure degree of pigmentation (250). A higher L* value represents lighter skin that contains less absorbing melanin (38). In the current study, skin color was measured at two sites: the upper inner arm which represents constitutive or genetically inherited skin color at a non-UV exposed site and the outer forearm which represents both constitutive and facultative (tanning) skin color (37). Hair was shaved from a small patch of skin (3 cm2) on the forearm using a disposable razor. Three consecutive readings were made at slightly different areas on each skin site. Natural pigmentation was reported to be best described in the L* vs b* correlation and a single variable, the Individual Typology Angle (ITA°) describing this correlation was derived using the following calculation: ITA° = ArcTangent{(L-50)/b)] x 1 8Oht. Skin color was classified using ITA° variables into the following categories: Very Light> 55 > Light > 41 > Intermediate> 28 > Tanned> 10> Brown > -30 > Dark (251). Skin color data was collected using a color data software (SpectraMagic NX, Konica Minolta Sensing, Inc.) and transformed into an excel file. 3.4.3 Blood collection and laboratory methods Blood sample collection was conducted at either the outpatient blood collection laboratory at the BC Children’s Hospital, Vancouver or by a certified phiebotomist if the test 66 was conducted at a community clinic. For each participant, a tube of non-fasting blood (—1 0 mL) was collected by venepuncture into a vacutainer containing sodium heparin as an anti-coagulant and placed on ice immediately. Blood samples were centrifuged within 2 hours (3000g, 10 minutes). Plasma was removed and stored at -80°C in Dr. Sheila Innis’s laboratory located at the Child and Family Research Institute, Vancouver for subsequent analyses. Samples underwent no freeze-thaw cycles and were stored up to 12 months before being analyzed for 250HD in February 2010. Plasma was delivered to the BC Biomedical Laboratories Ltd. on dry ice for analyses. Plasma 250HD was determined by the DiaSorin LIAISON® 25-OH Vitamin D TOTAL Assay, a competitive chemiluminescence immunoassay used for the quantitative determination of both 25OHD and 25OHD3 metabolites. According to the kit manufacturer, inter- and intra-assay % coefficient of variations were 3.2-8.5% and 6.9-12.7%, respectively. The assay had high sensitivity (analytical sensitivity: < 2.5 nmol/L; functional sensitivity: < 10 nmol/L) and specificity (25OHD 104%; 250HD3 100%). The measuring range was 10-309 nmol!L. Plasma PTH was determined by the DiaSorin LIAISON® N-TACT® PTH Assay using the same technology that recognized intact PTH only. The analytical sensitivity was 0.1 pmol/L. The measuring range was 0.1-212 pmol/L (252). BC Biomedical Laboratories Ltd. participates in the Vitamin D External Quality Assessment Scheme, an external quality control program for 25OHD measurement (253). 3.5 Data Analyses Statistical analyses were performed using SPSS Statistics 18.0 for Macintosh (SPSS Inc., Chicago, IL 2010). Data were checked for normality using histograms. A cutoff 67 of < 25 nmoL’L was used to indicate deficiency (52). Two commonly used cutoffs were used to define vitamin D insufficiency; 50 and 75 nmol!L (11, 12). The Mann-Whitney U test and Kruskal-Wallis test were used to determine whether the distribution of vitamin D intake was different across various factors. A one-way ANOVA was used to test whether or not the distribution of ITA° was differed by ethnicity. Estimates were considered statistically significant if P < 0.05 using the Bonferroni adjustment method. Because age, week of gestation, ethnicity, season, BMI, smoking, and vitamin D intake (including supplement use) have been reported to affect plasma 250HD concentrations, multiple regression models were used to examine the independent relationships between each of these variables and plasma 250HD concentration. Similarly, determinants of the prevalence of vitamin D insufficiency (< 50 and < 75 nmoIJL) were estimated using multivariate logistic regression. For the multiple regression analyses only, missing values were imputed by using the average for that variable. For example if a participant did not provide her age, the mean age of the entire sample was assigned to that participant. Estimates were considered statistically significant if P < 0.05 using the Bonferroni adjustment method. The normal reference range for PTH is less than 6.4 pmol/L; therefore in the current study, a plasma PTH level of greater than 6.4 pmol/L was used as a cutoff for hyperparathyroidism (254). The relationship between plasma 250HD and PTH concentrations was evaluated with linear and exponential regression models. The exponential regression line was used to determine if a threshold exists above which further increases in 250HD do not further suppress PTH concentration. To examine the effect of calcium intake on the relationship between PTH and 250HD, participants were divided into tertiles according to calcium intake (low, < 1029 mg/d; medium, 1029-1528 mg/d; and high,> 1528 68 mgld), and plasma 250HD concentration (low, < 55 nmol!L; medium, 55-74 nmolIL; and high, > 74 nmol/L). Multiple regression analysis was used with PTH as the dependent variable and calcium intake and plasma 250HD concentration as the independent variables, with and without an interaction term in the model. 3.6 Miscellaneous Each participant was provided with a $25 grocery voucher to cover her transportation cost. After data analyses were completed, a letter addressing the study summary, participant’s individual 250HD concentration, and recommendation on vitamin D intake was mailed to their addresses (Appendix H). 69Ñ CHAPTER 4: RESULTS 4.1 Recruitment and Participant Characteristics Between February 2009 and February 2010, 340 pregnant women were recruited to participate in the study. About half of participants (48%) were recruited from the BC Women’s Hospital and the rest were from The Healthiest Babies Possible Program (16%), Douglas College Prenatal Classes (26%), and the general community (10%), who contacted the investigator directly in response to advertisement in newspapers or on poster boards (Table 4.1). Of the 340 participants blood was obtained from only 336 due to difficulties in drawing blood from four participants. An average of 84 individuals were recruited in each of the winter (December 21 — March 20), spring (March 21 — June 20), summer (June 21 — September 22), and fall months (September 23 — December 20). Participants characteristics are given in Table 4.2. The majority of women (6 1%) were 30 years or older. The mean age of the women was 31 years and they ranged in age from 16-47 years. About 46% of participants were of European ancestry, 20% were Chinese, 9% were South Asians, and 25% were classified as Others. The “Other” category included Latin American (n = 21), Black (n 12), Filipino (n = 10), Southeast Asian (n = 9), Korean (n = 6), Japanese (n = 5), Iranian and Afghan (n = 4), Arab (n = 3), and other (n = 15). Participants were from Vancouver (52%), Richmond (18%), Burnaby (11%), and the remainder of the Lower Mainland (19%). The majority of women (66%) reported a normal pre-pregnancy BMI (18.5 —24.9 kg/rn2). Most participants did not smoke (94%) during their pregnancy. Almost all of the participants (93%) took vitamin D and/or calcium supplements (94%), either as a single supplement or as one of multiple supplements. Participants were generally well-educated with 68% having completed at least some university. Of those who 70 reported their family income (n = 213), only 18% had an income < $40,000 and 28% had a family income > than $120,000. 4.2 Sun Exposure Self-reported sun exposure, outdoor clothing practice, sunscreen use, sun bathing, and sun bed use are summarized in Tables 4.3-4.5. During the two months prior to the clinic visit, 78% of participants reported that they spent most of their time indoors during the work days. The majority of individuals (88%) spent less than 60 minutes/d outdoors. During leisure days, 31% of participants indicated that they were mostly indoors and 65% reported a mixture of time spent indoors and outdoors. About 53% spent less than 60 minutes/d outdoors and the rest spent 60 minutes/d or more outdoors (Table 4.3). Women reported that when they were outside they tended to wear clothes that covered their legs and arms, but not their head, face or hands (Table 4.4). About 19% traveled outside Canada in the two months prior to the clinic visit. As shown in Table 4.5, participants were more likely to use a sunscreen in the summer than other seasons. If a sunscreen was used, the most commonly chosen sun protection factor was 15 or 30. Most participants did not sunbathe or use sun beds regardless of the season. None of the participants used spray tanning on their arms. 4.3 Vitamin D Intake Vitamin D supplement use by age, ethnicity, season, and education is summarized in Table 4.6. Of the participants, 88% and 12% of participants had a total vitamin D intake (food plus supplements) 10 ig/d and < 10 pg/d, respectively. Median vitamin D intakes from diet and supplement by age, ethnicity, season and pre-pregnancy BMI are given in 71 Table 4.7. Median (1st quartile, 31X quartile) vitamin D intakes from diet, supplement, and diet and supplement were 5.5 (3.5, 7.7), 10.0 (10.0, 10.0), and 16.0 (13.1, 20.4) jig/d, respectively. The majority of supplement users took a prenatal multivitamin-mineral supplement containing 10 .tg of vitamin D3. Milk products (milk, chocolate milk and milk added to foods) were the main dietary sources of vitamin D. The median vitamin D intake from milk products was 3.2 (1.3, 5.2) ig!d. This amount is slightly more than 2.5 tg in a cup of milk, which is mandatorily fortified with vitamin D in Canada (8). Vitamin D intake from food alone did not differ by age (P = 0.295) and ethnicity (P = 0.978). Vitamin D intake from supplements was significantly higher among older participants (: 30 y; P = 0.032) and women of European ethnicity (P = 0.025). Total vitamin D intake did not differ by age (P = 0.160). Europeans had a significantly higher vitamin D intake from diet and supplements than non-Europeans [16.9 (13.3, 33.2) cf. 15.6 (13.0, 19.0) jig/d; P = 0.039]. Season and pre-pregnancy BMI did not have any effects on vitamin D intake from food, supplements and total intake. 4.4 Biochemical Outcome Measures Plasma 250HD concentrations were normally distributed (Figure 4.1). Percentiles of 250HD by age, week of gestation and ethnicity are shown in Table 4.8. Unadjusted mean plasma 250HD concentrations and prevalence of vitamin D insufficiency are shown in Table 4.9. Mean (95% CI) 250HD concentrations and the prevalence of vitamin D insufficiency based on two cutoffs adjusted for age, week of gestation, ethnicity, season, pre-pregnancy BMI, smoking status, and total vitamin D intake are summarized in Table 4.10. 72 Overall the mean (95% CI) 25OHD was 66.7 (64.2, 69.1) nmol/L. In multivariate analysis, ethnicity, season, and total vitamin D intake had a significant impact on 250HD concentrations. Women of European ethnicity had higher mean 25OHD concentrations than women of Other ethnicity [69.1 (62.8, 75.3) cf. 59.0 (52.2, 65.8) nmol/L; P = 0.004]. However, there were no differences between Europeans [69.1 (62.8, 75.3) nmol/L], South Asians [58.5 (48.6, 68.5) nmol!L], and Chinese [63.9 (56.1, 71.7) nmol/L]. Mean 250HD was lower in winter than spring [55.1 (47.7, 62.5) cf. 64.7 (57.7, 71.7) nmol/L; P = 0.035] and summer [55.1 (47.7, 62.5) cf. 67.4 (59.9, 74.9) nmol/L; P = 0.002] but not fall [63.2 (55.6, 70.8) nmol/L] (Figure 4.2). Mean 250HD concentrations by month were plotted and means were adjusted for ethnicity (Figure 4.3). Throughout the calendar year, 250HD concentrations generally rose from a low in January of 52.2 (44.9, 59.5) nmol/L to a peak in September of 86.4 (77.6, 95.3) nmol/L before falling through to the end of the year. Compared to January, mean 250HD concentrations were significantly higher in June [74.5 (67.2, 81.9) nmol/L; P = 0.003], July [77.4 (68.8, 86.0) nmol/L; P = 0.000] and September [86.4 (77.6, 95.3) nmol/L; P = 0.0001. However, the mean 250HD concentration in August [53.9 (47.4, 60.3) nmol/L] was lower than expected. Dietary vitamin D intake predicted 25OHD concentration, such that every 5 .tg increase in total vitamin D intake was associated with a 2.1 (1.1, 3.2) nmol/L increase in mean 250HD concentration (P = 0.000) (Table 4.11). As shown in Table 4.9 and Table 4.10, only 4 (1%) women had a 250HD concentration indicative of deficiency (< 25 nmol!L). However, 24% (19, 28) and 65% (60, 70) of participants were vitamin D insufficient based on cutoffs of 50 and 75 nmoIJL, respectively. Ethnicity, season, total vitamin D intake and pre-pregnancy BMI, but not age, week of gestation or smoking status were significant determinants of having insufficient vitamin D (< 73 50 nmol/L). South Asians and Others had a 34% (4, 64) (P = 0.018) and 21% (1, 41) (P = 0.032) greater prevalence of insufficiency than Europeans, respectively. The prevalence of insufficiency was significantly higher in winter (56%) than spring (27%) and fall (26%). Every 5 jig decrease in total vitamin D intake was associated with a 34% (12.5, 56) higher prevalence of insufficiency. Vitamin D insufficiency was 42% (11, 73) more prevalent among women with a BMI < 18.5 kg/rn2 compared to those with a normal BMI (18.5 — 24.9 kg/m2). Only ethnicity (P = 0.037) and total vitamin D intake (P = 0.041) were significant determinants of vitamin D status when the insufficiency cut-off of 75 nmol/L was used. Although ethnicity had an overall significant effect, there were no differences in 250HD among four ethnic groups. Each additional 5 jig of vitamin D was related to a 12.5% (0.5, 24.5) lower prevalence of insufficiency (< 75 nmol/L). Plasma PTH concentrations were normally distributed. Unadjusted and adjusted mean (95% CI) plasma PTH concentrations are shown in Table 4.12. Means were adjusted for age, week of gestation, ethnicity, season, pre-pregnancy BMI, smoking status, and 250HD concentration. Overall the mean (95% CI) PTH concentration was 3.4 (3.2, 3.6) pmol/L. Week of gestation, ethnicity, pre-pregnancy BMI and 25OHD concentration were significant determinants of PTH concentration. Women who were less than 27th week of gestation had significantly lower PTH than those ? 27th week of gestation [3.0 (2.5, 3.5) cf. 3.5 (3.0, 4.0) pmol/L; P = 0.0111. Women of South Asian ethnicity had higher PTH than women of Other ethnicity [3.8 (3.0, 4.6) cf. 2.8 (2.3, 3.3) pmol/L; P = 0.033]. Women with a BMI < 18.5 kg/rn2 had much lower PTH concentrations than those with a BMI? 30 kg/m2 [2.6 (1.8, 3.4) cf. 4.0 (3.2, 4.8) pmol/L; P = 0.042]. About 5% of women (n = 17) had PTH concentration greater than 6.4 pmol/L. 74 4.5 Relationship between 25-Hydroxyvmtain D and Parathyroid Hormone Concentrations The relationship between plasma 250HD and PTH concentrations is depicted in Figure 4.4. The linear regression line showed that PTH was only weakly inversely associated with 250HD with a slope of -0.01 and 3.4% of variation explained by the model (R2 = 0.034; P = 0.001). No inflection point was detected by the exponential regression model (R2 = 0.03; P = 0.00 1) meaning that a threshold did not exist above which further increases in 250HD did not further suppress PTH concentration. 4.6 25-Hydroxyvmtain D, Parathyroid Hormone and Calcium Intake Median (1st quartile, 3’ quartile) calcium intakes from diet, supplement, and total intake were 1238 (610, 1234), 500 (250, 375), and 1738 (920, 1749) mg/d, respectively. Figure 4.5 gives the mean (95% CI) plasma PTH for each tertile of plasma 250HD by tertile of dietary calcium intake. Calcium intake was significantly associated with plasma PTH (P = 0.026), independent of 250HD concentration. The lowest tertile of calcium intake (< 1029 mg’d) was significantly associated with higher PTH (P = 0.047). However, when 250HD concentration was added into the multiple regression model, neither 250HD (P = 0.192) concentration nor calcium intake (P = 0.641) was associated with PTH concentration after adjusting for 250HD concentration and calcium intake. There was no significant interaction between calcium intake and 250HD concentration with plasma PTH (P = 0.907). 4.7 Skin Color Exposed (outer forearm) and unexposed (upper inner arm) skin color classification 75 (ITA°), by ethnicity is shown in Table 4.13. Approximately 25%, 39%, and 24% of individuals had light, intermediate, and tanned exposed skin color, respectively (Figure 4.6). About 23% and 51% of participants had very light and light unexposed skin color, respectively (Figure 4.7). With regard to ethnicity, 95% of Europeans and 97% of Chinese had exposed skin color ranging from light to tanned whereas 95% of South Asians and 89% of Others had intermediate to brown exposed skin color. The majority of Europeans and Chinese had very light to intermediate unexposed skin color whereas most women of South Asia and Other ethnicity had light to brown unexposed skin color. The distribution of skin color indices (L, a, b and ITA° variables as described in Section 3.4.2) at upper inner arm and outer forearm by ethnicity are given in Table 4.14. A higher ITA° indicates lighter skin color. The mean ITA° was higher at the upper arm (45 ± 17) than the forearm (32 ± 16) (P = 0.000). Within each ethnicity, this trend remained; however, ITA° varied between ethnicities. For example, South Asians had a 48% lower upper arm ITA° than Europeans indicating naturally darker skin color (P = 0.000). Exposed skin color at the forearm was also darker by 51% (P = 0.000). As shown in Table 4.11, every 100 increase in ITA° at the upper inner arm and the forearm was significantly associated with a 5.0 (2.3, 7.7) nmol/L increase (P = 0.000) and 4.7 (1.8, 7.6) nmol/L decease (P = 0.001) in 250HD concentrations, respectively after adjusting for season, total vitamin D intake and ITA°. This indicates that lighter skin color at the upper arm (representing ‘constitutive’ or genetically inherited skin color) was related to a higher 250HD concentration whereas lighter skin color at the forearm (representing genetically inherited skin color and tanning) was linked to a lower 250HD concentration. Adjusted mean 250HD concentrations by skin color categories are shown in Table 4.15. These means were adjusted for season, total vitamin D intake, and 76 skin color categories. Brown and dark skin color categories were combined because only few people had dark skin. Once ITA° scores were collapsed into categories (very light, light, intermediate, tanned, brown and dark), the mean 250HD concentrations did not differ anymore at the forearm; however, unexposed skin color was still a strong determinant of 250HD concentration (P = 0.004). Women with very light and light skin had significantly higher 250HD than those with tanned skin: 72.5 (66.1, 78.9) nmol, 70.3 (65.2, 75.5) nmol/L, and 47.9 (37.8, 58.0) nmol/L, respectively. 77 Ta bl e 4. 1 Pa rti ci pa nt re cr u itm en t B rit ish Co lu m bi a W om en ’s H os pi ta l & H ea lth Ce nt re 16 3 (48 ) Th e H ea lth ie st B ab ie sP os sib le Pr og ra m 55 (16 ) D ou gl as Co lle ge Pr en at al Cl as se s 88 (26 ) Pa rti ci pa nt s co n ta ct ed th e in ve sti ga to r 34 (!O ) 1N um be ro fp ar tic ip an ts in st ud y (pe rce nta ge o f p ar tic ip an ts in st ud y) 2Pe rc en ta ge o f p ar tic ip an ts ap pr oa ch ed w ho v isi te d th es e sit es 3Pe rc en ta ge o f p ar tic ip an ts ar ro ac he d w ho ag re ed to pa rti ci pa te R ec ru ite d fro m n (% )‘ % A pp ro ac he d 2 % A gr ee d to pa rti ci pa te 3 - - 16 - - 50 — 30 - — 37 — 69 ‘ - - 35 N /A N /A — . 1 00 Table 4.2 Characteristics of participants All Age (years) <30 30 Week of gestation <27 >27 Ethnicity European Chinese South Asian Other’ Area of residence Vancouver Richmond Burnaby Other2 328-336 129 (39) 206 (61) 113 (34) 219 (66) 155 (46) 66 (20) 30(9) 85 (25) 169 (52) 60 (18) 36(11) 63 (19) 76(23) 89 (26) 92 (27) 79 (24) 19(6) 312 (94) All Vitamin D supplement Users Nonusers Calcium supplement Users Nonusers Pre-pregnancy BMI (kg/rn2) <18.5 18.5-24.9 25-29.9 >30 Education <High school High school Trade/vocational training University Family income per year <$40,000 $40,000-<80,000 $80,000-<120,000 $120,000 Don’t know Don’t want to say 322-336 311 (93) 25 (7) 316 (94) 20 (6) 22 (7) 213 (66) 64 (20) 23 (7) 5(1) 53 (16) 49(15) 228 (68) 38(11) 56 (17) 61(18) 59(18) 74(22) 48 (14) Characteristic n (%) Characteristic n (%) Season3 Winter Spring Summer Fall Smoking status Smokers Nonsmokers BMJ, Body Mass Index ‘Latin American 21(6%); Other 15 (4%); Black 12 (4%); Filipino 10 (3%); Southeast Asian 9 (3%); Korean 6 (2%); Japanese 5 (1%); Iranian and Afghan 4 (1%); Arab 3 (1%) 2Surrey, Delta, New Westminster, Coquitlam, North Vancouver, Kelowna, Fort St. James, Bella Coola, Abbotsford, Port Moody, Hope, White Rock, Langley, Castlegar, Bowen Island, Kamloops 3’Winter’ months: December21 - March 20; ‘Spring’ months: March 21 - June 20; Sunimer’ months: June 21 - September 22; ‘Fall’ months: September 23 - December 20 79 Table 4.3 Time spent outdoors during work days and leisure days Time outdoors Work days Leisure days (mm/day) n (%) n (%) <15 94(28) 27(8) 15-<30 122 (36) 59(18) 30-<60 79 (24) 90 (27) 60-<120 29 (9) 91 (27) 120-240 8 (2) 56 (17) >240 4(1) 13(4) Table 4.4 Outdoor clothing practice over the past two months Parts of the body coveredFrequency n (%) 2Head Face Hands Legs Arms Never 147 (44) 303 (90) 228 (68) 14 (4) 22 (7) Sometimes 126 (38) 27 (8) 92 (27) 78 (23) 107 (32) Often 23(7) 3(1) 9(3) 54(16) 56(17) Frequently 24 (7) 1 (0) 5 (1) 62 (18) 84 (25) Always 16 (5) 2 (1) 2 (1) 128 (38) 67 (20) ‘Pantyhose, long dresses, trousers 2long sleeves 80 Table 4.5 Sunscreen use, sun bathing, and sun bed use by season Characteristic Winter’ Spring’ Summer’ Fall’ n (%) n (%) n (%) n (%) All Sunscreen use Never Sometimes Most of the time Always Sun protection factor Didn’t usually use a sunscreen <15 15 30 >30 Don’t know Parts of the body covered Face Amis Legs 3Other 76 (23) 39 (51) 19 (25) 8(11) 10 (13) 36 (47) 5 (7) 22 (29) 7 (9) 4 (5) 2(3) n = 272 23 (85) 1 (4) 0 (0) 3(11) 89 (26) 30(34) 30(34) 18 (20) 11(12) 31(35) 3 (3) 27 (30) 18 (20) 7 (8) 3 (3) 2 n =62 42 (68) 0 (0) 0 (0) 20(32) 92 (27) 15 (16) 26 (28) 32 (35) 19 (21) 15 (16) 3 (3) 35 (38) 28 (30) 10(11) 1 (1) 2 n = 77 36 (47) 4 (5) 0 (0) 37 (48) 79 (24) 33 (42) 24 (30) 12 (15) 10 (13) 24 (30) 1 (1) 26 (33) 16 (20) 7 (9) 5 (6) 2 n = 54 34(63) 0 (0) 0 (0) 20 (37) Sun bathing No Yes Use of sun beds No Yes 74 (97) 2 (3) 74 (97) 2(3) 80 (90) 9(10) 89 (100) 0 (0) 79 (86) 13 (14) 89 (97) 3 (3) 75 (95) 4 (5) 77 (97) 2(3) “Winter’ months: December 21 - March 20; ‘Spring’ months: March21 - June 20; Summer’ months: June 21 - September 22; ‘Fall’ months: September 23 - December 20 2Number of participants who answered this question. 3Face, arms, legs combinations or other parts of the body (e.g. neck, chest, hands, feet, shoulders) 81 Table 4.6 Vitamin D supplement use by age, ethnicity, season, and education Characteristic n (%) All 311 (93) Age (years) <30 115(89) 30 195 (95) Ethnicity European 139 (90) Other’ 172 (95) Season2 Winter 70 (92) Spring 82 (92) Summer 85 (92) Fall 74 (94) Education <High school 3 (60) High school 51(96) Trade/vocational training 43 (88) University 213 (93) ‘Chinese, South Asian, Latin American, Black, Filipino, Southeast Asian, Korean, Japanese, Iranian and Afghan, Arab, and other 2’Winter’ months: December21 - March 20; ‘Spring’ months: March 21 - June 20; Summer’ months: June 21 - September 22; ‘Fall’ months: September 23 - December 20 82 T ab le 4. 7 V ita m in D in ta ke (bi g/d ay )f ro m di et an d su pp le m en tb y ag e, et hn ic ity ,s ea so n , an d bo dy m as s in de x (B M I) M ed ia n (1s t q u il e 3rd qu ar til e) Su pp le m en t A ge (ye ars ) A ll Ch ar ac te ris tic D ie t - D ie t & su pp le m en t 5. 5( 3.5 ,7. 7) 10 .0 (1 0.0 ,10 .0) 16 .0 (1 3.1 ,20 .4) < 30 5.8 (3. 8, 8.2 )a 10 .0 (10 .0, 1O .0) a 15 .6 (13 .0, 19 .3) a ?3 0 5.2 (3. 4, 7. 6)a 10 .0 (10 .0, 1 2 •5) b 16 .1 (13 .1, 21 .3 )a Et hn ic ity Eu ro pe an 5.3 (3. 4, 8.2 )a 10 .0 (10 .0, 15 .9) a 16 .9 (13 .3, 22 .2 )a O th er ’ 5. 6 (3 8 7 7 )a 10 .0 (10 .0, 10 .0 )” 15 .6 (13 .0, 19 .0) ” Se as on 2 W in te r 5. 6 (4. 0, 9.0 )a 10 .0 (10 .0, 10 .0) a 15 .9 (13 .1, 20 .4 )a Sp rin g 6.3 (3. 5, 8.2 )a 10 .0 (10 .0, 10 .0) a 16 .3 (12 .8, 20 .6 )a Su m m er 4. 7 (3. 1, 73 )a 10 .0 (10 .0, 12 .5) a 15 .6 (13 .0, 20 .9 )a Fa ll 5. 6 (3. 9, 7. 8)a 10 .0 (10 .0, 12 .5) a 16 .3 (14 .0, 19 .6) a Pr e- pr eg na nc y BM I( kg /rn 2) < 18 .5 4. 6 (3. 2, 93 )a 10 .0 (10 .0, 10 .0) a 17 .0 (13 .7, 24 .3 )a 18 .5 -2 4. 9 5. 4 (3. 6, 7. 6)a 10 .0 (10 .0, 12 .5) a 16 .6 (13 .1, 20 .0 )a 25 -2 9. 9 6. 4 (3. 9, 8.5 )a 10 .0 (10 .0, 10 .0) a 15 .8 (13 .2, 20 .1 )a ?3 0 4. 8 (3. 3, 77 )a 10 .0 (10 .0, 10 .0) a 14 .0 (12 .2, 19 .3) a Es tim at es w ith in a co lu m n su bg ro up n o ts ha rin g a co m m o n su pe rs cr ip tl et te ra re sig ni fic an tly di ffe re nt , P < 0. 05 . ‘ Ch in es e, So ut h A sia n, La tin A m er ic an ,B la ck ,F ili pi no ,S ou th ea st A sia n, K or ea n, Ja pa ne se ,I ra ni an an d A fg ha n, A ra b, an d o th er 2’W in te r’ m o n th s: D ec em be r2 1 - M ar ch 20 ;‘ Sp rin g’ m o n th s: M ar ch 21 - Ju ne 20 ;‘ Su m m er ’m o n th s: Ju ne 21 - Se pt em be r2 2; Fa ll’ m o n th s: Se pt em be r 2 3 - D ec em be r2 0 T ab le 4. 8 Pe rc en til es o f p la sm a 25 -h yd ro xy vi ta m in D by ag e, w ee k o f g es ta tio n an d et hn ic ity Pe rc en til es o f 2 5- hy dr ox yv ita m in i D (nm ol/ L) Ch ar ac te ns itc n 1st t1 1 1 0 t h 2 5 t h 5 0 t h 7 5 t h 9 0 t h 9 5 t h A ll 33 2- 33 6 24 34 40 50 64 79 96 10 8 A ge (ye ars ) < 30 12 9 19 .0 31 .0 38 .6 47 .0 63 .0 77 .0 97 .2 10 4. 8 3 0 20 6 25 .2 37 .3 42 .0 51 .0 64 .5 80 .0 95 .5 10 8. 0 W ee k o fg es ta tio n < 27 11 3 24 .6 31 .6 40 .2 49 .0 61 .0 76 .0 95 .8 10 6. 8 ?2 7 21 9 25 .0 35 .9 40 .8 51 .0 66 .0 80 .5 96 .2 10 8. 0 Et hn ic ity Eu ro pe an 15 5 27 .7 39 .0 45 .0 55 .5 70 .0 85 .5 10 5. 2 10 9. 0 O th er ’ 18 1 24 .0 31 .0 38 .0 47 .0 60 .0 75 .0 88 .0 98 .0 ‘ Ch in es e, So ut h A sia n, La tin A m er ic an ,B la ck , F ili pi no , S ou th ea st A sia n, K or ea n, Ja pa ne se , I ra ni an an d A fg ha n, A ra b, an d o th er Age (years) Week of gestation <27 >27 Ethnicity concentrations and prevalence of insufficiency Plasma 25-hydroxyvitamin D (nmolJL) European Chinese South Asian 2Other Season3 155 66 30 85 72.1 (68.5, 75.7) 65.3 (59.2, 71.5) 59.6 (51.3, 67.9) 60.2 (56.0, 64.4) 14 (9, 20) 26(15,37) 40 (21, 59) 33 (23, 43) 57 (49, 65) 67 (55, 78) 77(61,93) 75 (66, 85) Pre-pregnancy BMI (kg/rn2) Smoking status Smokers Nonsmokers 19 312 66.4 (52.8, 80.0) 66.8 (64.3, 69.3) 26 (5, 48) 23 (18, 27) 68 (45, 91) 65 (59, 70) CI, Confidence Interval; BMI, Body Mass Index ‘Insufficiency <50 nmolJL and <75 nmol/L 2Latin American, Black, Filipino, Southeast Asian, Korean, Japanese, Iranian and Afghan, Arab, and other 3’winter’ months: December 21 - March 20; ‘Spring’ months: March 21 - June 20; Summer’ months: June 21 - September 22; ‘Fall’ months: September 23 - December 20 Table 4.9 Plasma 25-hydroxyvitamin D All Characteristic Mean Prevalence % (95% CI) n (95% CI) <50’ <75’ <30 30 322-336 66.7 (64.2, 69.1) 129 206 65.4 (61.1, 69.7) 67.4 (64.4, 70.3) 24 (19, 28) 27(19, 35) 21(16, 27) 26 (17, 34) 22 (16, 27) 113 64.9 (60.4, 69.5) 219 67.7 (64.8, 70.7) 65 (60, 70) 70 (62, 78) 63 (56, 69) 72 (63, 80) 62 (55, 68) Winter 76 59.5 (54.7, 64.3) 37 (26, 48) 74 (64, 84) Spring 89 67.4 (63.0, 71.8) 19(11, 27) 62 (52, 72) Summer 92 70.6 (65.3, 75.9) 23 (14, 32) 62 (52, 72) Fall 79 68.1 (63.2, 73.0) 16 (8, 25) 65 (54, 75) <18.5 22 57.7 (48.5, 66.9) 50 (27, 73) 77 (58, 96) 18.5-24.9 213 67.9 (64.8, 71.0) 18 (13, 24) 63 (57, 70) 25-29.9 64 68.6 (62.7, 74.6) 25 (14, 36) 61 (49, 73) 30 23 64.5 (55.3, 73.7) 26 (7, 46) 70 (49, 90) 85 Table 4.10 Adjusted mean plasma 250HD concentrations and prevalence of insufficiency Plasma 250HD (mnol/L) Characteristic Adjusted mean Adjusted prevalence % (95% CI) n (95% CI) < 50’ < 751 Age (years) Week of gestation <27 >27 Ethnicity European Chinese South Asian 2Other Season3 Winter Spring Summer Fall BMI (kg/rn2) <18.5 18.5-24.9 25-29.9 >30 Smoking status Smokers Nonsmokers 113 219 155 66 30 85 22 213 64 23 19 312 61.3 (54.8, 67.9)a 63.9 (57.1, 70.6)a 69.1 (62.8, 753)a 63.9 (56.1, 717)ab 58.5 (48.6, 685)ab 59.0 (52.2, 658)b 55.6 (45.2, 66.1)a 64.8 (59.1, 70.4)a 66.6 (59.3, 74.0)a 63.4 (52.9, 73.8)a 63.3 (52.6, 74.0)a 61.9 (58.0, 65.8)a 36(18, 53)a 35 (17, 53)a 20 (8, 32)a 31(12, 51)ab 54 (27, 80)b 41(23, 59)b 56 (36, 76)a 2711,43)b 36 (16, 56)ab 26 (8, 43)b 64 (39, 89)a 22 (10, 33)b 31 (l3,48) 29 (4, 55)ab 38 (9, 68)a 33 (23,42)a 79 (68, 91)a 70 (55, 84)a 64 (49, 79)a 72 (56, 88)a 80 (64, 97)a 80 (68, 92)a 82(71, 94)a 69 (53, 84)a 70 (54, 86)a 76 (62, 91)a 82 (64, 99)a 73 (61, 84)a 69 (53, 85)a 74 (54, 95)a 75 (54, 96)a 75 (67, 83)a <30 129 61.9 (55.3, 68.5)a 39 (21, 57)a 78 (66, 90)a ?30 206 63.3 (56.7, 69.9)a 32 (16, 49)a 71 (57, 85)a 76 55.1 (47.7, 62.5)a 89 64.7 (57.7, 717)b 92 67.4 (59.9, 749)b 79 63.2 (55.6, 708)ab 25OHD, 25-hydroxyvitamin D;CI, Confidence Interval; BMI, Body Mass Index Estimates adjusted for age, week of gestation, ethnicity, season, pre-pregnancy BMI, smoking, and total vitamin D intake. Estimates within a column subgroup not sharing a common superscript letter are significantly different, P <0.05. ‘Insufficiency <50 nmol/L and <75 nmol/L 2Latin American, Black, Filipino, Southeast Asian, Korean, Japanese, Iranian and Afghan, Arab, and other 3’Winter’ months: December 21 - March 20; ‘Spring’ months: March 21 - June 20; Summer’ months: June 21 - September 22; ‘Fall’ months: September 23 - December 20 86 Table 4.11 Estimated difference in 250HD according to selected variables Estimated differenceVariable 95% CI Pin 250HD (nmol/L) Total vitamin D intake’ Per 5 tg increase 2.1 (1.1, 3.2) 0.000 ITA°, per 100 increase2 Upper inner arm Forearm 250HD, 25-hydroxyvitamin D; CI, Confidence Interval ‘Estimates adjusted for age, week of gestation, ethnicity, season, pre-pregnancy Body Mass Index, and smoking 2Estimates adjusted for season, total vitamin D intake, and upper arm and forearm ITA° ITA°, Individual Typology Angle = ArcTangent[(L-50)/b)] x 1 80/it 5.0 (2.3, 7.7) 0.000 -4.7 (-7.6, -1.8) 0.001 87 Table 4.12 Mean plasma parathyroid hormone concentrations Unadjusted mean Age (years) 322-3 36 3.4 (3.2, 3.6) 129 206 3.4 (3.1, 3.6) 3.4(3.2, 3.7) 3.2 (2.7, 37)a 3.3 (2.8, 3.8)a Week of gestation Smoking status Smokers 19 Nonsmokers 312 CI, Confidence Interval; BMI, Body Mass Index Estimates adjusted for age, week of gestation, ethnicity, season, pre-pregnancy BMI, smoking, and 250HD concentration. Estimates within a column subgroup not sharing a common superscript letter are significantly different, P <0.05. ‘Latin American, Black, Filipino, Southeast Asian, Korean, Japanese, Iranian and Afghan, Arab, and other 2’Winter’ months: December 21 - March 20; ‘Spring’ months: March 21 - June 20; Summer’ months: June 21 - September 22; ‘Fall’ months: September 23 - December 20 All Characteristic n (95% CI) (95% CI) <30 30 Adjusted mean 3.2 (2.8, 3.7) <27 113 3.0 (2.7, 3.4) 3.0 (2.5, 3•5)a 27 219 3.6(3.1,3.8) 3.5(3.0,4.0)” Ethnicity European 155 3.3 (3.0, 3.5) 3.1 (2.6, 3.5) Chinese 66 3.5 (3.1, 4.0) 3.3 (2.7, 39)ab South Asian 30 4.2 (3.4, 5.0) 3.8 (3.0, 4.6)a Other’ 85 3.2 (2.9, 3.5) 2.8 (2.3, 3.3)” Season2 Winter 76 3.7 (3.2, 4.1) 3.4 (2.8, 4.0)a Spring 89 3.3 (3.0, 3.7) 3.3 (2.8, 39)a Summer 92 3.3 (2.9, 3.7) 3.3 (2.7, 3.8)a Fall 79 3.3 (2.9, 3.7) 3.1 (2.5, 37)a Pre-pregnancy BMI (kg/rn2) <18.5 22 2.8 (2.2, 3.3) 2.6 (1.8, 3•4)a 18.5-24.9 213 3.4(3.1,3.6) 31(2736)aI 25-29.9 64 3.4 (3.0, 3.8) 3.2 (2.7, 38)ab ?30 23 4.1 (3.3, 5.0) 4.0 (3.2, 4.8)” 2.6 (1.7, 3.4) 3.0 (2.1, 3g)a 3.4 (3.2, 3.6) 3.5 (3.2, 3.8)a 88 T ab le 4. 13 Ex po se d (ou ter fo re ar m ) a n d u n ex po se d (up pe r i nn er ar m )s ki n co lo rc la ss ifi ca tio n by et hn ic ity IT A ° Cl as sif ic at io n’ A ll Eu ro pe an Ch in es e So ut h A sia n O th er 2 A lln (% ) 33 6 15 5 (46 ) 66 (2 0) 30 (9 ) 85 (2 5) Ex po se d sk in co lo ur V er y lig ht n (% ) 8 (2) 8 (5) 0 (0) 0 (0) 0 (0) Li gh tn (% ) 84 (25 ) 60 (39 ) 17 (26 ) 1 (3) 6 (7) In te rm ed ia te n( % ) 13 1 (39 ) 60 (3 9) 35 (53 ) 5 (17 ) 31 (36 ) Ta nn ed n (% ) 82 (24 ) 26 (17 ) 12 (18 ) 15 (50 ) 29 (34 ) B ro w n n (% ) 28 (8) 1 (1) 2 (3) 9 (30 ) 16 (19 ) D ar kn (% ) 3( 1) 0( 0) 0( 0) 0( 0) 3( 4) U ne xp os ed sk in co lo ur V er yl ig ht n( % ) 76 (2 3) 58 (3 7) Li gh tn (% ) 17 1 (51 ) 77 (50 ) In te rm ed ia te n (% ) 47 (14 ) 16 (10 ) Ta nn ed n (% ) 29 (9) 3 (2) B ro w nn (% ) 10 (3 ) 1( 1) D ar k n (% ) 3 (1) 0 (0) 1IT A 0, In di vi du al Ty po lo gy A ng le A rc Ta ng en t[( L- 50 )/b )] x 18 0/ it Cl as sif ic at io n o f I TA ° v ar ia bl es : V er y lig ht > 55 ° > Li gh t> 41 ° > In te rm ed ia te > 28 ° > Ta nn ed > 10 ° > B ro w n> - 30 ° > D ar k 2L at in A m er ic an ,B la ck ,F ili pi no ,S ou th ea st A sia n, K or ea n, Ja pa ne se ,I ra ni an an d A fg ha n, Ar ab ,a n d o th er 13 (2 0) 0( 0) 5( 6) 44 (6 7) 10 (3 3) 40 (4 7) 7( 11 ) 8( 27 ) 16 (1 9) 2( 3) 9( 30 ) 15 (1 8) 0( 0) 3( 10 ) 6( 7) 0( 0) 0( 0) 3( 4) 0O Ta bl e 4. 14 D ist rib ut io n o fv ar ia bl es o fu n ex po se d (up pe ri nn er ar m )a n d ex po se d (ou ter fo re ar m )s ki n co lo r b y et hn ic ity Et hn ic ity Sk in sit e a2 b3 1T A 0 4 A ll U pp er ar m 67 ±6 * 6± 2 16 ±3 45 ±1 7 Fo re ar m 62 ±6 8± 2 19 ±2 32 ±1 6 Eu ro pe an U pp er ar m 70 ± 3 5 ± 1 15 ± 2 54 ± 7 Fo re ar m 65 ±4 8± 2 18 ±3 39 ±1 1 Ch in es e U pp er ar m 68 ± 2 5 ± 1 16 ± 2 48 ± 6 Fo re ar m 63 ±4 7± 2 19 ±2 35 ±1 0 So ut h A sia n U pp er ar m 60 ± 5 7± 2 19 ± 2 28 ± 14 Fo re ar m 57 ±5 9± 1 20 ±2 19 ± 13 O th er 5 U pp er ar m 63 ± 8 7 ± 2 18 ± 3 32 ± 23 Fo re ar m 59 ±7 9± 2 20 ±2 22 ±1 9 * M ea n± SD “2’3C om m is si on In te rn at io na ld e PE cl ai ra ge L* a* b* sy ste m :L v al ue re pr es en ts re la tiv e br ig ht ne ss o fc o lo r ( bla ck to w hi te ); a v al ue ca pt ur es sk in re dn es s; b v al ue m ea su re s pi gm en ta tio n 4IT A O , I nd iv id ua lT yp ol og y A ng le = A rc Ta ng en t[( L- 50 )/b )] x 18 0/ it 5L at in A m er ic an ,B la ck ,F ili pi no ,S ou th ea st A sia n, K or ea n, Ja pa ne se ,I ra ni an an d A fg ha n, A ra b, an d o th er Table 4.15 Adjusted mean 250HD concentrations by skin color classification 1 Adjusted mean 250HD (nmol/L)ITA° Classification fl (95% CI) Exposed skin colour Very light 8 55.2 (38.3, 72.0)a Light 84 57.9 (49.9, 65.9)a Intermediate 131 57.3 (50.9, 63.6)a Tanned 82 66.1 (60.1, 72.1)a Brown and dark 31 73.9 (64.4, 83.4y’ Unexposed skin colour Very light 76 72.5 (66.1, 78.9y’ Light 171 70.3 (65.2, 755)a Intermediate 47 640565716)ab Tanned 29 479(378580)b Brown and dark 13 55.6 (39.2, 721)ab 250HD, 25-hydroxyvitamin D; hA0, Individual Typology Angle; CI, Confidence Interval Estimates adjusted for season, total vitamin intake and skin colour categories. Estimates within a column subgroup not sharing a common supercript letter are significant different, p < 0.05. ‘Very light>55°>Light>4 1 °>Intermediate>28°>Tanned>1 0°>Brown>-3 0°>Dark 91 40— 30— 0 20- I Z 10- 150 200 25-hydroxyvitamin D (nmol/L) Figure 4.1 Histogram of plasma 25-hydroxyvitamin D concentration 92 ba b It aN I 80 - 70 - 60 - 50 - 40 - 30 20 10 0 ab Winter Spring Summer Season Fall Figure 4.2 Mean (95% CI) plasma 25-hydroxyvitamin D concentrations by season, adjusted for age, week of gestation, ethnicity, pre-pregnancy body mass index, smoking, and total vitamin D intake. Estimates not sharing a common superscript letter are significantly different (P <0.05). * ON 100 90 80 70 60 50 40 30 20 10 0 Jan Feb Mar Apr May Jun Jul Aug Sep (32) (27) (27) (34) (28) (31) (23) (42) (21) Month (n) Oct Nov Dec (25) (37) (9) Figure 4.3 Mean (95% CI) plasma 25-hydroxyvitamin D concentrations by month, adjusted for ethnicity. *Mean 25-hydroxyvitamin D concentrations in June, July and September were significantly lower than in January (P <0.05). 93 14 Figure 4.4 Association of plasma parathyroid hormone with 25-hydroxyvitamin D concentration. The slopes of the linear (y = -0.0142x + 4.3291; R2 = 0.0341) and exponential regression lines (,, 38965e-O.0041x; R2 = 0.03) are shown. 94 12 10• 0 0 0 0 8 0 0 C C C C I Linear - - - Exponential 0 0 0 0 0 0 0 6 0 0 0 0 0 0 00 0 8 8 0 0 4. 2 0- 0 0 0 0 0 e 0 00 0 od’ 0 0 00 0000 0O? e0 0 0 00 0 50 100 150 25-hydroxyvitamin D (nmol/L) 200 5- , — ‘ 4. 5- o 4 E 3 .5 U 3 2. 5 2 0 1. 5- 1- 0. 5- 0- 25 -h yd ro xy vi ta m in D (nm olJ L) < 1 02 9 • 10 29 -1 52 8 D >1 52 8 Fi gu re 4. 5 M ea n (95 % CI )p la sm a pa ra th yr oi d ho rm on e co n ce n tr at io n by te rt ile o fp la sm a 25 -h yd ro xy vi ta m in D co n ce n tr at io ns an d te rt ile o f c al ci um in ta ke (m g/d ). T I < 55 55 -7 4 > 74 Figure 4.6 Distribution of study participants by exposed skin color Figure 4.7 Distribution of study participants by unexposed skin color 140 120 80 60 C a) £ 20 0 z 0 Skin Color Category 180 160 - 140- . 120 • 100 0 40 20 Z 0 I— Skin Color Category 96 CHAPTER 5: DISCUSSION AND CONCLUSIONS In this chapter, study findings are discussed in relation to reports in the literature on vitamin D intake from diet and supplements, vitamin D status based on plasma 250HD concentration and prevalence of deficiency and insufficiency in pregnant women as well as women of reproductive age, and factors (season and ethnicity) affecting 250HD concentration. The relationships between plasma 250HD and PTH concentrations and skin color are also discussed. Study limitations, areas for future research and conclusions are outlined. 5.1 Vitamin D Intake Vitamin D intakes of the pregnant women in this study are comparable to or higher than intakes of most other reports of women’s intakes, whether pregnant or not, and whether considering intakes from food alone or from the combination of food and supplements (28, 255, 256). In the present study, only 4% of women did not meet the Al of 5 ig/d and 12% had a total vitamin D intake less than 10 rigId. However, most experts indicate that the current Al set in 1997 is not high enough to maintain appropriate vitamin D status (15). For example, the Canadian Pediatric Society has recommended that pregnant women receive 50 Ig/d of vitamin D, an amount 10 times the current recommendation (13). If this recommendation was used to evaluate intakes of women in the current study, only 1% of them (n =4) received? 50 tg of vitamin D from food and supplements. Median dietary vitamin D intake from food in our study [5.5 (3.5, 7.7) jig/d] is somewhat higher than that reported for women of reproductive age (19-50 y; n = 5018) in the CCHS (2.2) [3.5 (1.7, 6.0) .tgId] (28). However, when compared to British Columbia 97 females, our participants had similar vitamin D intake from foods. British Columbia females aged 19-30 and 31-50 had mean ± SD intakes of 5.8 ± 0.9 jig!d and 4.7 ± 0.4 jig/d, respectively. About 40% and 66% of women in these two age groups did not meet the Al from food alone (255) compared with 46% in our study. Milk products were the main source of vitamin D in both the CCHS (all women of reproductive age) and our study contributing to 56% (2.0 jig/d) and 58% (3.2 jig/d) of dietary vitamin D intake, respectively (28). US data from NHANES 2005-2006 showed lower average vitamin D intakes from food alone: 3.6 ± 0.3 and 4.4 ± 0.3 jig/d for women 19-30 y and 3 1-50 y, respectively (257). While women in our study have vitamin D intakes from food alone comparable to or higher than non-pregnant women of reproductive age in North America, vitamin D intakes from food of our participants are also comparable to or slightly higher than previously reported intakes among pregnant women. For example, the daily mean ± SD vitamin D intake from food was 4.2 ± 3.6 jig for women living in Northern Canada (106). In a large US pregnancy cohort (n = 1543), vitamin D intakes from food alone during the 1 and 21 trimester were 5.4 ± 2.9 and 5.8 ± 2.9 jig/d, respectively (156). Vitamin D intake from dietary supplements must be included to accurately estimate the total vitamin D intake. Without the inclusion of supplements, mean nutrient intakes are underestimated (258). In our study, vitamin D supplements provided a large contribution to maternal vitamin D intake. Over 90% of participants took one or more vitamin D containing supplements with a median intake of 10 j.tg/d. Vitamin D intake from supplements and total intake in our study are higher than that reported in other studies of non-pregnant women of reproductive age and pregnant women. In the British Columbia Nutrition Survey, 34% (25, 44) of women 19-30 y had taken a supplement containing 98 vitamin D in the past month with a median intake of 6.6 jig/d from supplements. For females 31-50 y, the prevalence of vitamin D supplement use was 43% (36, 50), with a median intake of 7.0 jig/d (256). Vitamin D supplement intake information from CCHS 2.0 is not yet available. Supplement use is also common among US non-pregnant women. For example, in the NHANES 2005-2006, 34% of women aged 31-50 y reported using vitamin D containing supplements with a mean ± SD intake of 7.9 ± 0.3 jig/d (257). In a group of US pregnant women (n = 2251), vitamin D intakes from supplements and total intake were 5.5 and 10.3 ± 0.1 jig!d, respectively. However, the percentage of women reported using vitamin D supplements during pregnancy was not reported (155). It appears that pregnant women are more likely to take vitamin D containing supplements than non-pregnant women. Analyses of the NHANES 2001-2006 data revealed that among pregnant women taking vitamin D supplements, the median vitamin D dose was 10 jigld and 90% were taking 10 jig or more as compared to 85.5% in our study. In their study, pregnant women in the first trimester were more likely to take vitamin D supplements compared to non-pregnant women (61% and 32%, respectively; P < 0.00 1). Pregnant women were more likely to be taking any vitamin D supplement with increasing trimester (6 1%, 72%, and 86%, respectively; P for trend = 0.002) (259). Similar results were reported in black and white Americans living in Pittsburgh (n = 400). Ninety percent of women in their last trimester of pregnancy reported multivitamin or prenatal vitamin use at least once per week and only 45% reported supplement use in the periconceptional period (26). While the use of vitamin D containing supplements is common in North America, the frequency of consumption appears to be much lower in pregnant women in other countries. For example, only one third of pregnant women used dietary supplements in New Zealand (224). In a New Zealand 99 pregnancy study (n = 439), the total vitamin intake was much lower with a median intake of 2.1 jigld. The lower vitamin D intake is expected because vitamin D fortification is not mandatory in New Zealand and supplementation is less common (224). In a group of Irish pregnant women (n = 43), none of the women took a supplement that contained vitamin D (115). The percentage of women who used vitamin D containing supplements did not differ by ethnicity in our study; however, in some studies, non-Europeans were less likely to use prenatal multivitamins and other dietary supplements that contained vitamin D (155, 259). In the present study, ethnicity had a significant impact on total vitamin D intake. In our study, Europeans had a 1.3 jig higher daily mean vitamin D intake than non-Europeans (P = 0.039). This difference was likely to be attributable to Europeans’ higher intakes from supplements (P = 0.025) because vitamin D intakes from food alone did not differ significantly between two groups. Although the difference between ethnic groups reached statistical significance, a difference of 1.3 jig is likely of little clinical significance. Other studies have also indicated an effect of ethnicity on vitamin D intake. In NHANES 1999-2000, total vitamin D intake was much lower in non-Hispanic black females of reproductive age (19-50 y) and Mexican Americans (6.1 ± 0.4 and 5.7 ± 0.4 jig, respectively) than non-Hispanic whites (7.8 ± 0.4 jig) (157). Similarly, data from the CHMS showed that Canadians in other racial groups consumed significantly less fortified milk, which is the main dietary source of vitamin D, than did those classified as White (74). The total vitamin D intake of native pregnant women (8.1 ± 5.5 gg/d) was significantly lower than that of non-native women (13.2 ± 5.9 jig/d). Lower intakes of native women were related to limited availability of vitamin D fortified foods in remote communities (106). 100 5.2 Vitamin D Status and Prevalence of Inadequacy It appears that vitamin D status of pregnant women in this study was similar to or better than that of other reports of pregnant and non-pregnant women. The overall mean 250HD in our study [66.7 (64.2, 69.1) nmolJL] is very similar to that reported for women of reproductive age in the CHMS [69.5 (65.8, 73.2) nmol!Lj. In the present study, only 1% of women had a 250HD concentration indicative of deficiency (< 25 nmollL). Below 25 nmollL, the risk of rickets and osteomalacia increases substantially (52). However, the 250HD concentration associated with optimal maternal and infant health remains unknown. Recent evidence suggests that at least 75 nmol/L may be required for optimal health (13, 30). Despite a median intake of 16.0 ug/d of vitamin D from food and supplements, 24% (19, 28) and 65% (60, 70) of participants in this study were vitamin D insufficient based on cutoffs of 50 and 75 nmol/L, respectively. The prevalence of inadequacy in our study is very similar to that reported in the CHMS, which found that 10% (6, 15) and 64% (56, 71) of women of reproductive age had 250HD concentrations below 37.5 and 75 nmol/L, respectively (74). Since non-white women made up a larger percentage of women in our study (54%) than the CHMS (18%), it is difficult to compare our results to theirs. When vitamin D status of only white women (Europeans) are compared, women in our study [69.1(62.8, 75.3) nmol/L] had lower mean 250HD concentration than that reported in the CHMS [755 (71.5, 79.6) nmolJL]; however, the difference is not statistically significant. Most of the women in our study were taking vitamin D containing supplements and although supplement consumption was not reported in the CHMS, vitamin D supplement use is probably less frequent in women in the CHMS. Despite the supplement use in our study, 250HD concentrations were still similar to those reported in the CHMS. It is important to note that the CHMS also used 101 the DiaSorin LIAISON® 25-OH Vitamin D TOTAL Assay to detect 250HD concentration (74). Among US women 13-44 y (n = 5173), the mean serum 25OHD concentration was 59 (57, 61) nmol/L. Overall, 10% (8, 12), 42% (38, 46), and 78% (76, 80) of women had 25OHD concentrations <25, <50, and < 75 nmoIJL, respectively. This group of women had a lower mean 250HD and higher prevalence of vitamin D deficiency and insufficiency compared to our sample possibly due to a lower percentage of supplement users (32%) compared to our sample (93%) and a greater proportion of black women (259). When compared with the few studies of pregnant women in Canada, our participants appear to have better vitamin D status possibly due to higher supplement use and living at a lower latitude (49°N). In the Inuvik zone (68°N), the mean plasma 25OHD concentrations of pregnant women (4th — 32nd week of gestation) were 59.8 ± 29.4 nmol/L for Caucasians (n = 33), 52.1 ± 25.9 nmol/L for Native Indians (n = 37), and 50.1 ± 19.3 nmol/L for Inuits (n = 51) (106). Among 50 white pregnant women living in Newfoundland (47°N) and Labrador (52°N), 2%, 42%, and 80% had 25OHD concentrations <25, <50, and < 75 nmol!L. A few reasons may explain their worse vitamin D status. St John’s (47°N), the capital city of Newfoundland, is one of the foggiest, snowiest and cloudiest cities in Canada. Endogenous vitamin D production is very minimal under these conditions. In addition, the authors suggested that Newfoundland residents are less likely to consume enough vitamin D fortified food (109). Although vitamin D inadequacy was more prevalent in other Canadian studies, findings are consistent indicating that vitamin D insufficiency is common in Canadian pregnant women. Data from the NHANES 2001-2006 revealed that US pregnant women (n = 928) also had a high prevalence of vitamin D insufficiency. The mean (95% CI) serum 25OHD concentration was 65 (6 1-68) nmol/L, which is similar to ours. Vitamin D 102 deficiency (< 25 nmol!L) was found in 7% of their subjects compared to 1% in our study. About 33% and 69% of women were insufficient in vitamin D based on cutoffs of 50 and 75 nmol/L. The higher prevalence of vitamin D deficiency in their study is possibility due to a lower percentage (72%) of supplement users and a greater proportion (16%) of Blacks (259). In other countries, the prevalence of vitamin D inadequacy is higher than that seen in Canada and the US. For example in France (46°N), 34% of pregnant women (n = 59) had 250HD < 25 nmol/L with a mean ± SD concentration of 35.5 ± 21 nmoli’L in winter (116). In a multi-ethnic (European, Afircan, Maori, Middle Eastern, and Polynesian) study conducted in New Zealand (41°S; n = 90), 61% and 87% of pregnant women had 250HD concentrations below 25 and 50 nmol!L, respectively (126). Possible reasons for higher prevalence of low vitamin D status in these other countries may include having fewer options for vitamin D in foods and limited supplement use. For example in New Zealand, there is voluntary fortification of margarine and fluid milk, the latter which is fortified at a much lower level of 1 ‘g per 250 mL than in Canada where 2.5 jig are added to 250 mE (260). There are also many other factors that could affect their vitamin D status, such as environmental conditions (i.e., latitude, season and climate) and lifestyle (i.e., sun avoidance and sunscreen use). 5.3 Factors Associated with 25-Hydroxyvitamin D Concentrations Ethnicity was a determinant of 250HD concentration but the magnitude of the effect was less pronounced than in other studies possibly due to higher supplement use. Women of European ethnicity had a significantly higher 250HD than women of Other ethnicity [69.1 (62.8, 75.3) cf. 59.0 (52.2, 65.8) nmol!L]. When compared with Europeans, South Asians and Others were 34% and 21%, respectively, more likely to be vitamin D 103 insufficient (< 50 nrnolfL). The likely explanation for observed differences by ethnicity in our study is reduced cutaneous vitamin D synthesis in darker skinned compared to fairer skinned individuals as it does not appear that vitamin D intake is markedly lower in non-Europeans [15.6 (13.0, 19.0) ig/d] versus Europeans [16.9 (13.3, 22.2) jig/d] (19). Another explanation could be that non-Europeans, for example Asian women, are more likely to cover up or avoid the sun (75). A similar effect of ethnicity on 250HD concentration has been reported in many studies. For Canadian women 20-39 y in the CHMS, the mean 250HD was higher in whites than non-whites [75.5 (71.5, 79.6) cf. 48.9 (46.5, 51.2) nmol/L; P < 0.05] (74). Although in our study, Europeans also had higher 250HD than non-Europeans [69.0 (62.8, 75.3) cf. 60.5 (54.3, 66.7) nmol/L; P = 0.0011, the magnitude of the difference is not as large as the CHMS. In pregnant women living in Northern Canada (68°N), native mothers tended to have lower concentrations of 250HD. The mean difference between native and non-native mothers was approximately 10 nmol/L (106). In NHANES 2001-2006, non-Hispanic white women in the third trimester of pregnancy had much higher 250HD concentrations (93 nmolIL) than non-Hispanic black (45 nmol/L) or Hispanic (69 nmol/L) women (P < 0.001). The prevalence of vitamin D insufficiency (< 75 nmolJL) was much higher in non-Hispanic black (95%) and Hispanic (83%) than non-Hispanic white women (54%) (259). In white and black US pregnant women (40°N; n 400), only 5% of white women had 250HD concentrations below 37.5 nmol/L whereas 29% of black women did (26). In addition to studies in North America, ethnicity is also an important determinant of vitamin D status in studies conducted in other populations, for instance Pakistani immigrant and Norwegian pregnant women living in Norway (59°N; n = 76) (120) as well as western, Turkish and Moroccan women residing in Netherlands (52°N; n = 358) (122). 104 Season was also a determinant of vitamin D status with a lower mean 250HD in winter [55.1 (47.7, 62.5) nmol/LJ than spring [64.7 (57.7, 71.7) nmollL] and summer [67.4 (59.9, 74.9) nmollLj. The prevalence of insufficiency (< 50 nmol/L) was about 30% higher in winter than spring and fall. The mean 250HD concentration in August was lower than expected possibly due to that over 83% of women used sunscreen, at least sometimes. The magnitude of the effect of season appears less pronounced than in other studies; again, perhaps because of supplement use. Data from the CHMS showed that women of childbearing age had significantly lower mean (95% CI) 250HD concentration in winter [64.2 (59.7, 68.8) nmol/Lj than summer [74.3 (67.5, 81.1) nmol/Lj (74). A study of pregnant women (n = 593) living in Newfoundland (47°N) and Labrador (52°N) has also reported lower 250HD in winter (52.1 nmolfL) than summer (68.6 nmol/L); however, the difference was not statistically significant. In winter, 89% of women were vitamin D insufficient (< 75 nmol/L) whereas in summer, only 64% were insufficient (110). Studies in other countries have reported similar findings on the influence of season on vitamin D status. The median (1st 3” quartile) 250HD was significantly lower in winter and spring [36.5 (25.3, 46.3) nmol/L] than summer and fall [47.3 (32.3, 46.3) nmol/L] among Greek pregnant women (38°N; n 123) (118). In a cohort of pregnant Irish (52°N; n = 43), the mean ± SD 250HD concentration in the winter period was significantly lower than in the summer (32.2 ± 12.2 cf. 50.9 ± 23.4 nmol/L, respectively). The prevalence of vitamin D deficiency (< 25 nmoL/L) was 30% in winter compared to 17% in summer. About 91% and 58% of women had 250HD concentrations below 50 nmol/L in winter and summer, respectively (115). Total vitamin D intake had a significant impact on vitamin D status, though of lesser magnitude than ethnicity or season. Every additional 5 jig of vitamin D was associated with a 105 2.1 nmol/L increase in 250HD concentration. It is important to note that very few women (7%) did not take supplements in our study and of those who took supplements, most were taking 10 ug/d which may have made it difficult to get a true estimate of the effect of vitamin D intake on 250HD concentration as compared to if there were fewer supplement users and/or more variability in supplement dose. A similar effect of vitamin D intake on 250HD concentration has been reported in other studies of pregnant women. In Canadian mothers living in the Inuvik zone, each additional 5 pg of vitamin D from supplements resulted in a 7.2 nmol/L increase in mean 250HD concentration, which is higher than that of our study (106). Both vitamin D supplementation dose and duration of vitamin D supplement use were associated with higher 250HD concentrations in US pregnant women (259). Among women not taking vitamin D containing supplements, mean 250HD concentrations by increasing trimester were 46, 46, and 54 nmol/L, respectively, compared to 61, 69, and 84 nmol/L among women taking supplements. Later trimester was associated with longer duration of vitamin D supplement use. The mean 250HD concentration was about 25 nmol/L higher in third trimester (median supplement duration was 304 days) compared to first-trimester women (61 days) even though the vitamin D doses were similar. The authors suggested that 250HD concentrations could be improved during early pregnancy by initiating vitamin D supplementation before pregnancy to increase supplementation duration (259). Although participants in the current study had a median vitamin D supplement intake of 10 ig/d, this dose may not be adequate for raising all women’s 250HD concentration to ? 75 nmolIL, particularly for higher-risk women, such as ethnic minorities and during the winter season. Prior vitamin D supplementation randomized trials in pregnant women concluded that although 10 jig (234), 20 jig (235), or 25 jig/d (186) of vitamin D raised 250HD during 106 pregnancy, these doses were not enough to raise the concentration above 75 nmol/L. In the present study, although the mean 250HD did not differ by pre-pregnancy BMI, vitamin D insufficiency (< 50 nmol/L) was 42% more prevalent among women with a BMI < 18.5 kg/m2 compared to those with a normal BMI (18.5 — 24.9 kg/rn2). The difference may be a result of a smaller sample size (n = 22) and an uneven distribution of ethnicity in the BMI < 18.5 kg/m2 group as over 85% of women were non-Europeans. Previous studies have shown that obesity is associated with lower vitamin D status (101) possibly due to excessive deposition of vitamin D in the adipose tissue and limited sun exposure caused by limited mobility (20, 77, 100). However, we were unable to detect this relationship, perhaps due to the fact that only 20% and 7% of women were classified as ‘overweight’ (BMI 25-29.9 kg/rn2) and ‘obese’ (BMI 30 kg/rn2), respectively. Also, since in our study pre-pregnancy weight was self-reported, there may be a tendency to under-report weight especially among overweight or obese people (261). Pregnancy causes an increase in plasma volume, which begins to rise at 6 - 8th week of gestation and increases to about 45% by 30 weeks. Increased plasma volume and red cell mass, and a decrease in serum albumin may affect circulating concentrations of 250HD in various stages of pregnancy (262). In our study, the effect of week of gestation (<27 and 27th week) on 250HD concentrations was not significant. Studies that examined this relationship have found inconsistent results. Some suggested that 250HD concentrations should be equal or even decrease during pregnancy (169). In contrast, Bodnar et al (26) reported higher 250HD concentrations in late(37th — 42nd week of gestation), as compared to early (4t — 21 week of gestation), pregnancy. In the NHANES 200 1-2006, serum 250HD concentrations were also higher with later trimester. The authors suggested that the higher 107 concentrations later in pregnancy may be a result of the longer duration of vitamin D supplementation because for those women not taking supplements, 250HD concentrations were similar throughout pregnancy (259). 5.4 Effect of Skin Color In the present study, vitamin D status was associated with ethnicity. However, ethnicity is only a proxy measure for skin color, which varies considerably within racial categories. Melanin in skin acts as a natural sunscreen; therefore, to obtain the same amount of vitamin D, a darker skinned individual requires a longer period of UVB exposure than does a fairer skinned individual (19, 83). Our research is relatively novel in that few studies have looked for the association between quantitative measures of skin color and 250HD concentration. Skin color measurements at the upper arm and forearm are valid measures of constitutive (natural) and facultative (tanned) skin color, respectively (263). Higher ITA° indicates lighter skin color. In the current study, every 100 increase in ITA° at the upper arm or forearm was significantly associated with a 5 (2.3, 7.7) nmol/L increase and 4.7 (1.8, 7.6) nmol/L decease in 250HD concentrations, respectively. The results indicate that lighter constitutive (genetically inherited) skin color was related to higher 250HD concentrations. Conversely, lighter facultative (genetically inherited and tanning) skin color (indicating less sun exposure) after adjusting for constitutive skin color was associated with lower 250HD concentrations. Our findings are consistent with others. In one study, the unexposed skin L* value (a higher L* value represents lighter skin that contains less UV absorbing melanin) was positively associated with baseline 250HD concentrations and in order to increase 250HD 108 concentrations by 30 nmolJL, a lighter skinned individual required a lower UV dose (73). A study conducted among young adult Canadians (n = 107) found that people with darker constitutive skin had lower serum 250HD concentrations (P = 0.033), when measured in winter (15). While these two studies only measured unexposed skin color, both exposed and unexposed skin color were measured in a group of Pacific Islanders (n = 87) and Europeans (n = 255) living in New Zealand. It was found that each 100 increase in ITA° at the forearm (facultative skin color), a marker of tanning or sun exposure, was associated with a 5 nmolJL decrease in 250HD (P < 0.00 1). In contrast to our results, they did not find any relationship between constitutive skin color and 250HD concentration. The authors suggested that the unexpected result may be due to a lower proportion of darker skinned people included in their study (37). 5.5 Relationship between 25-Hydroxyvitamin D and Parathyroid Hormone Concentrations In the present study, week of gestation, ethnicity, pre-pregnancy BMI and 250HD concentration were significant determinants of plasma PTH concentration. A lower mean PTH concentration was found in early (< 27th week of gestation) as compared to late pregnancy 27 week of gestation). Similarly as reported in some studies, PTH falls during the first and second trimester to the low-normal range compared with non-pregnant women, and may increase back to the mid-normal range during the third trimester (264, 265). We found that South Asian women had a higher mean PTH than women of Other ethnicity [3.8 (3.0, 4.6) cf. 2.8 (2.3, 3.3) pmol/L]. As shown in other reports, PTH levels may vary by ethnicity. In one study, postmenopausal African American women (50-75 y; 11=208) 109 had higher PTH than white American women (266). We also found that obese women (BMI 30 kg/rn2)had significantly higher PTH concentrations than underweight women (BMI 18.5 kg/rn2): 2.6 and 4.0 pmol/L, respectively. This result is similar to that of others (267, 268). For example, Kamycheva et al (267) reported that in a Norwegian population (n 7954), serum PTH was positively related to BMI in both genders (P < 0.00 1). The highest quartile of PTH (> 4.2 prnol/L) was a significant predictor for obesity (P < 0.001) when adjusted for age, physical activity and serum calcium. Since abnormal calcium metabolism (269) and low vitamin D status (101) have been linked to obesity, one would expect the same to be true with PTH as it is related to both calcium and vitamin D. The relationship between obesity and PTH may be explained by several mechanisms. In response to a low serum calcium level, PTH stimulates the renal hydroxylation of 250HD to 1 ,25(OH)D, which in turn elevates the calcium influx into adipocytes (269). Increased intracellular calcium promotes lipid storage in fat tissues. However, these studies were conducted in mice, not humans (270). Another explanation is that the elevated PTH in obesity might be the result but not the cause of obesity. A disturbed renal handling of calcium in obesity leads to negative calcium balance and thus elevated PTH levels (271). Secçndary hyperparathyroidism, due to vitamin D insufficiency, may lead to accelerated bone loss (55). Hyperparathyroidism was present in 5% of our participants (PTH > 6.4 pmol/L). It is difficult to compare our results to those of others partly due to different assays used to determine PTH concentration and cutoffs used to define hyperparathyroidism. Few studies have reported rates of hyperparathyroidism in pregnant women. For example, in a multi-ethnic study of pregnant women (23’ 32nd week of gestation; n = 971) living in Sydney, Australia, 4% of women, which was similar to 5% in our study, had secondary 110 hyperthyroidism; however, the cutoff for hyperthyroidism used in this study was 5 pmol/L (272). In a study conducted in women of child-bearing age living in Beijing and Hong Kong (20-35 y; n = 441), hyperparathyroidism was defined as a serum PTH concentration greater than 7.6 pmol/L, the upper limit of the reference range provided by the kit manufacturer. About 24% and 2% of women had hyperparathyroidism in Beijing and Hong Kong, respectively, compared to 5% in our study (273). As reported in some studies, PTH concentration is inversely associated with 250HD up to a threshold, above which it plateaus at a minimum level (32). However in the present study, 250HD and PTH was only weakly inversely associated with no apparent threshold. Maximal suppression of PTH has been used to determine sufficiency for 250HD in some studies. The threshold for 250HD at the point of maximal suppression of PTH has varied by study ranging from 30 (57) to 90 mnol/L (58). This wide range of estimates may be due to the influence of calcium intake, different assays, population characteristics, and statistical models used to define the relationship between PTH and 250HD (56). A group of African American women (50-75 y; n = 208) were randomly assigned to a vitamin D supplement of 20 j.tg/d or a placebo for two years. The vitamin D dose was increased to 50 tg/d for the final study year. Both groups received calcium supplements (1200-1500 mg/d). PTH was inversely related to 250HD (r = -0.23; P < 0.01) and the threshold was found to be 40-50 nmol/L, 44 nmol/L, or> 40 nmol/L using Loess, Line-Line model, or exponential decay model, respectively. However, the rates of bone loss did not differ between people with 250HD concentration above or below 40 nmolJL (266). In South East Asian women of child-bearing age (18-40 y; n = 504), the threshold was found to be 52 nmol/L (274); however, among women of child-bearing age living in Beijing and Hong Kong (20-35 y; n = 111 441), no threshold was detected possibly due to a lack of high 250HD concentrations in the sample (273). In a study of US children and adolescents (n = 735), the 250HD threshold for PTH suppression was 98.5 nmol/L; however, when high PTH concentrations (> 10.6 pmol/L) were excluded, no point of inflection was detected (275). Based on a systematic review, Aloia et al (266) reported that most threshold estimates clustered between 40 and 50 nmol/L or 70 and 80 nmolJL. In order to estimate two thresholds simultaneously, a three-phase approach was developed by Durazo-Arvizu et al (276) recently. The three phases were: 1) PTH concentrations decline rapidly at a constant rate with increasing concentrations of 25(OH)D; 2) the rate of decrease in PTH concentrations with increasing concentrations of 25(OH)D changes to a slower rate; and 3) PTH is maximally suppressed. In US men and women 65-87 y (n = 387), the model identified two thresholds of 30 nmol/L and 70 nmol/L, which were similar to those reported by Aloia et al (266). The authors concluded that the three-phase model might be superior to the two-phase approach as it simultaneously estimated the two threshold clusters; however, validation of the model in other data sets is required (276). Few studies have examined the relationship between 250HD and PTH in pregnant women. For example, in an observational study of 374 Caucasian and South East Asian women, there was a statistically significant inverse relationship between 250HD and PTH at < 16th week of gestation (r = -0.18; P < 0.001) and 28th — 32nd week of gestation (r = -0.15; P = 0.003); however, the threshold was not determined in their study (33). Although a threshold for 250HD could be identified in most studies mentioned above, it was not detected in the current study and it is generally agreed that PTH suppression should not be used to recommend optimal vitamin D status because PTH can be influenced by many other factors and from a biological perspective, maximal PTH suppression may not be the 112 “optimal” state at all parts of the lifespan (49, 266, 275). 5.6 Effect of Calcium Intake on 25-Hydroxyvitamin D and Parathyroid Hormone The PTH suppressing effect of 250HD has been found to differ with calcium intake (266); therefore, in our study, dietary calcium intakes were considered in the model of 250HD and PTH. When 250HD concentration was added to the regression model, neither 250HD concentration nor calcium intake was associated with PTH. There was no interaction between calcium intake and 250HD with PTH. Our results differ from that of Steingrimsdottir et al (36), who reported, in an Icelandic adult population (n = 2310), at lower calcium intakes a higher 250HD concentration was required to cause maximal suppression of PTH. The effect of calcium intake has also been examined in Asian populations. Similar to our findings, studies conducted among non-pregnant women of reproductive age living in Jakarta (n = 126), Kuala Lumpur (n = 378), Beijing (n = 220), and Hong Kong (n = 221) have shown that the relationship between 250HD and PTH did not differ between women with different calcium intakes (273, 274). The authors suggested that it might be that calcium intakes (mean intake at 657 mg/d) amongst women in their study were not high enough to suppress PTH (274). Since in the current study, the median calcium intake was quite high at 1738 mg/d, there may be other explanations. Although no influence of calcium intake on the relationship between 250HD and PTH was found, lower calcium intakes (< 1029 mgld) were associated with higher PTH, independent of 250HD. This result is consistent with others (36, 277). For instance, Steingrimsdottir et a! (36) reported that the lowest tertile of calcium intake (< 800 mg/d) was significantly associated with higher serum PTH (P = 0.04). 113 5.7 Limitations This study has several limitations. A convenience sample of pregnant women was used so that the results can not be generalized to Canadian pregnant population as a whole or Vancouver pregnant women because non-white women made up a greater proportion (over 50%) of women in our study than in the Canadian population. However, the mean age of women in our study was 31 y, which is not markedly different from the average age (29.9 y) of women giving birth in British Columbia in 2007 (278). Recmitment bias may have occurred as women taking vitamin D containing supplements may have been more likely to take part in the study than those who didn’t use supplements. Further, the majority of women in our study sample were well-educated and had high family income. It is likely that these women were of above average socio-economic status. Whether the women in our study have higher or lower plasma 250HD concentrations than other women is not known. Since the demographics of pregnant women living in Vancouver and the Lower Mainland have not been reported in Canadian census or other reports, we could not compare the characteristics of our participants to all pregnant women living in these areas. Due to the nature of the cross-sectional study design, we are limited in our ability to make causal inferences as data were collected from one time point only. However, a cross-sectional design was an appropriate method used to achieve our goal which was to determine vitamin D status of pregnant women. Women were not eligible to participate in the study if they had any co-morbid conditions such as gestational diabetes, cardiac or renal disease, and conditions associated with vitamin D malabsorption. These conditions, if any, were self-reported and we did know whether the participants included in the study were indeed free of these diseases or 114 conditions. However, they should have accurately reported their health conditions because these pregnant women visited their doctors on a regular basis; therefore, their doctors would have informed them if they had any diseases or conditions listed above. Although we measured height, pre-pregnancy weight was self-reported. A semi-quantitative FFQ was used to assess nutrient intake. Self-reported dietary intake can be of limited validity or accuracy; however, our FFQ was specifically designed to measure calcium and vitamin D intake and has been validated in a group of multi-ethnic Canadian young adults during winter. In their study, each participant completed the FFQ twice (repeated for reproducibility assessment) and the FFQ results were highly correlated (r = 0.663; P < 0.00 1). The FFQ results were also highly correlated with 7-day food dairy results and with serum 250HD concentrations (r = 0.529; P < 0.001; r = 0.481; P < 0.001, respectively) (249). One of the cutoffs for vitamin D insufficiency used in our study was 75 nmol/L; however, the exact level required for optimal health remains controversial. Further, the evidence base for optimal 250HD in pregnancy is lacking. Some researchers have suggested an optimal value as high as 100 nmol/L (12) and if this was used as the cutoff in our study, 92% of women would be classified as vitamin D insufficient. 5.8 Directions for Future Research Current research on the relationship between maternal vitamin D status and infant birth weight has produced inconsistent results. The trials that exist have tended to be small and involved women with very low vitamin D status (222, 225). Further observational studies are required to determine the association between birth weight and vitamin D status. 115 Therefore, after all the participants in our study give birth to their babies, we will access their infant’s birth weight from the BC Perinatal Registry Database and will investigate this relationship as soon as this data is available. At present, we have no clear guidelines about how much vitamin D women should take or evidence to confirm whether current guidelines are adequate for optimal health of mother and child. In order to determine optimal vitamin D intakes for pregnant and lactating women, dose-response studies are needed, for example, maternal and infant responses to maternal vitamin D supplementation during pregnancy and lactation. Outcome variables may include maternal and infant 250HD concentrations, breastmilk vitamin D content, fetal and infant growth, maternal and infant PTH, and bone biomarker concentrations. Several investigators have examined the dose response between 250HD and supplemental vitamin D intake in non-pregnant adults (148, 279); however, these studies should not be extrapolated to pregnant and lactating women as physiological changes in pregnancy and lactation alter the absorption, metabolism and uptake of some nutrients, especially calcium (262). In a systematic review commissioned by the US Office for Dietary Supplements rated all intervention trials with vitamin D during pregnancy as being of “low methodological quality”. The authors concluded that further research in this area is needed due to a lack of controlled clinical trials (280). With the knowledge of the dose-response between vitamin D intake and 25OHD concentration, studies on functional outcomes are needed to find out what cutoffs should be and to establish vitamin D requirements during pregnancy and lactation. Large randomized control trials are needed to determine if vitamin D supplementation reduces preeclampsia and other adverse pregnancy outcomes. However, randomized trials can be expensive and sometimes unethical to conduct among pregnant and lactating women. 116 5.9 Conclusions We examined the vitamin D status in 336 pregnant women living in Vancouver and the Lower Mainland. Despite a median intake of 16 jig/d of vitamin D from food and supplements, 24% and 65% of women were vitamin D insufficient based on 250HD cutoffs of 50 and 75 nmol/L indicating that the current Al for pregnant women is not enough to raise all women’s 250HD above 75 nmol/L. Fortunately, only 1% of women had 250HD concentrations below 25 nmol/L. Compared to other studies, women in our sample had higher 250HD concentrations, which may reflect the greater availability of fortified foods such as fluid milk and margarine in Canada. Additionally, over 90% of women were taking vitamin D containing supplements. Ethnicity, skin color, season, and total vitamin D intake were determinants of vitamin D status. Europeans had higher 250HD concentrations than Others. Ethnicity is only a proxy measure for skin color, so that skin color was measured. Lighter skin color at the upper arm (constitutive) was related to higher 250HD concentrations whereas lighter skin color at the forearm (facultative) was linked to lower 25OHD concentrations. The mean 25OHD was lower in winter than spring and summer. Total vitamin D intake was positively related to 250HD. Week of gestation, ethnicity, pre-pregnancy BMI, and 250HD concentration were significant determinants for PTH. Plasma PTH was weakly inversely related to 250HD with no apparent inflection point. There was no significant effect of dietary calcium intake on the relationship between 25OHD and PTH; however, lower calcium intakes were associated with higher PTH concentrations. Most of our vitamin D is obtained through skin synthesis by the action of UVB light (6). Unfortunately in Vancouver, Canada (49°N) there is not enough sunlight in the winter months to make sufficient vitamin D. The current advice about 10-20 minutes of daily sun 117 exposure during the summer months does not increase 250HD concentration substantially, while sufficient sun exposure that could achieve a benefit would compromise skin health (281). Also women with darker skin or who cover up when outside tend to have lower vitamin D levels. It would seem safer and more effective to increase vitamin D intake from food; however, it is not likely that women will change dietary patterns to increase vitamin D intake, for example, consume more animal organs or fatty fish. Increasing consumption of other sources such as eggs and fortified margarine is conflicted with the current perceptions of healthy eating due to their high cholesterol and/or fat content (28). Although encouraging increased consumption of fortified milk appears to be an option, milk may be less consumed in some cultures (i.e. Chinese) and it will be difficult to obtain enough vitamin D solely from milk. 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Vitamin D intake to attain a desired serum 25-hydroxyvitamin D concentraiton. Am J Clin Nutr 2008;87:1952-8. 280. Cranney A, Horsley T, O’Donnell 5, Weiler HA. Effectiveness and safety of vitamin D in relation to bone health. Evidence-Based Report/Technology Assessment No. 158 Ottawa;2007. 281. Diffey BL. Modelling the seasonal variation of vitamin D due to sun exposure. Br J Dermatol 2010;doi:1O.l11l/j.1365-2133.2010.09697. 140 APPENDICES Appendix A: University of British Columbia Ethics Approval UBC C&W Research Ethics Board — S CHILDREN’S & WOMEN’S HEALTHA2-136, 950 West 28th Avenue CENTRE OF BRITISH COLUMBIA T3 (604)875-2496 A’ A OF T11 PROVUCAL HEAL,- FRCES )fFOwTVE I cwrebcw bc ca Website: http://wwwcfri cs/research_support> Research Ethics ETHICS CERTIFICATE OF MINIMAL RISK APPROVAL: AMENDMENT PRINCIPAL INVESTIGATOR: DEPARTMENT: UBC C&W NUMBER: -. IUBC/Land and Food Systems/Human rn John Green I .. H08-01447 and Animal Nutrition INSTITUTION(S) WHERE RESEARCH WILL BE CARRIED OUT: institution I Site UBC Vancouver (excludes UBC Hospital) Children’s and Women’s Health Centre of BC (md. Sunny Hill) Child & Family Research Institute fancouver Coastal Health (VCHRI/VCHA) Vancouver Community )ther locations where the research will be conducted: e may set up clinics in Community Centers around Vancouver and Richmond if necessary DO-INVESTIGATOR(S): ‘Jangyang Li Sheila M. lnris susan I. Barr tarbara Crocker SPONSORING AGENCIES: UBC Faculty of Land and Food Systems - “Vitamin D status and determinants in a multi-ethnic group of pregnant women living in Greater ‘ancou. - ROJECT TITLE: (ITAMIN D STATUS OF PREGNANT WOMEN IN VANCOUVER REMINDER: The current UBC Children’s and Women’s approval for this study expires: July 14, 2010 MENDMENT(S): MENDMENT APPROVAL DATE: Document Name I Version I Date May 18, 2010 dvertisements: Jew Study Brochure for Thank-You Letter 1 May 4, 2010 (itamin D Recruitment Brochure New Study 1 May 10, 2010 uestionnaire, Questionnaire Cover Letter, Tests: Supplement Form 1 May 4, 2010 Participant Thank You 3 May 17, 2010 ERTlFlCATlON: n respect of clinical trials: 1. The membership of this Research Ethics Board complies with the membership requirements for Research Ethics Boards refined in Division 5 of the Food and Drug Regulations. 2. The Research Ethics Board carries out its functions in a manner consistent with Good Clinical Practices. 3. This Research Ethics Board has reviewed and approved the clinical trial protocol and informed consent form for the trial Nhich is to be conducted by the qualified investigator named above at the specified clinical trial site. This approval and the iiews of this Research Ethics Board have been documented in writing. The amendment(s) for the above-named project has been reviewed by the Chair of the UBC Children’s and Women’s Research Ethics Board and the accompanying documentation was found to be acceptable on ethical grounds for research involving human subjects. Approved by one of: Dr. Marc Levine, Chair Dr. Caron Strahlendorf, Associate Chair 141 Appendix B: Children’s & Women’s Health Center of British Columbia Ethics Approval CHILDREN’S & WOMEN’S HEALTH Room A2-136, 950 West 28th Avenue ____ CENTRE OF BRITISH COLUMBIA Vancouver, BC V5Z 4H4 Phone: 604-875-3103 Fax: 604-875-2496 Research Review Committee Notice of Hospital Review PRINCIPAL INVESTIGATOR Department Certification Number Innis, Sheila Pediatrics CWO8-0158 CO-INVESTI GATORS: Innis, Sheila M.; Crocker, Barbara; SPONSORING AGENCIES: BC Medical Services Foundation; CW DEPARTMENTS, PROGRAMS AND ADMINISTRATIVE JURISDICTIONS IMPACTED BY THIS STUDY Pathology and Laboratory Medicine; Diagnostic/Ambulatory Program; ‘PROJECT TITLE VITAMIN D STATUS OF PREGNANT WOMEN IN VANCOUVER The Committee has reviewed the protocol for your proposed study, and has withheld issuing a Certificate of Approval until the following conditions have been satisfied or information provided: Please highlight or underline changes to consent form(s) or letter(s) and submit only one copy (IF APPLICABLE). Pmvide other requested information. Provisos: Pending receipt of a copy of the UBC Clinical Research Ethics Board Certificate of Approval - The signature of the Program Director, of the Pathology and Laboratory Medicine Program is required. The signature of the Medical Director and Program Director, Diagnostic/Ambulatory Program Program is required. In addition, under BCWH guidelines, the signature of the President BCWH is required. 142 Appendix C: Consent Form THE UNIVERSITY OF BRITISH COLUMBIA r Faculty of Land and Food Systems _______ r I fr Food, Nutñtlon and HealthSuite 230 2205 East MaYancouvaj B.C. Canada V6T 1Z4 iN: 604.822.2502 Fas: 604.822.6143 wwwjandfcod.ubc.ca PARTICIPANT INFORMATION AND CONSENT FORM VITAMll D STATUS OF PREGNANT WOMEN IN VANCOUVER Principal Investigator: Dr Tim Green Food, Nutrition & Health Land and Food Systems The University of British Columbia Co-Investigators: Dr Sheila Innis Department of Paediatrics Faculty of Medicine The University of British Columbia Ms Barbara Crocker Community Nutritionist Vancouver Coastal Health Authority Dr Susan Barr Food, Nutrition & Health Land and Food Systems The University of British Columbia Wangyang Li (graduate student) Food, Nutrition & Health Land and Food Systems The University of British Columbia 143 1. BACKGROUND Vitamin D is best known for its role in helping promote healthy bones. However, it has other important roles as well. Vitamin D is important throughout life but is particularly important during pregnancy. Some vitamin D is obtained from our diet; however, we get most of our vitamin D from exposing our skin to the sun. Anything that influences the amount of sunlight reaching skin such as season (winter versus summer), clothing covering skin, and skin color will affect vitamin D status. You are being invited to take part in a study that will help determine if pregnant women in BC are receiving enough vitamin D during pregnancy. 2. WHO IS CONDUCTING THE STUDY? The study is being conducted by the Nutrition Research Program of the Child and Family and Food, Nutrition, and Health Department of Land and Food Systems, University of British Columbia in collaboration with Public Health Dietitians of Vancouver Coastal Health Authority. The study is being funded by the “University of British Columbia Vitamins Research Fund”. 3. WHAT IS THE PURPOSE OF THE STUDY? • To determine the vitamin D status of pregnant women living in Vancouver. • To determine what factors (such as diet and sunlight exposure) influence the vitamin D status of pregnant BC women 4. WHO CAN PARTICIPATE IN THE STUDY? To be eligible to participate, you must be pregnant and near the end of the second or early third trimester to participate. 5. WHAT DOES THE STUDY INVOLVE? The study will be conducted at the Child and Family Research Institute (CFRI), located on the site of the Children’s and Women’s Hospital or at a community site. The procedures will take about 30-60 minutes of your time. 1. You will be asked to complete a confidential questionnaire that ask some general information about your health and lifestyle, your usual dietary habits, and your pregnancy. At any time you may refuse to provide any information that you do not feel comfortable sharing. 2. Your height and weight will be recorded. 3. Your skin color measured at two sites; your forearm and upper inner arm. Your hair will be shaved from a small patch of skin on the forearm. A disposable razor will be used to shave an area about 3 square centimeters. 144 4. A tube of blood (about 2 tablespoons) will be taken by a registered technologist or nurse. This blood sample will be used to measure your blood levels of vitamin D, parathyroid hormone, and other nutritionally related blood markers. 5. We will access your baby’s gender, date of birth, growth measurement for infant and mother, method of delivery, gestational age at delivery, gravity, and parity. 6. WHAT ARE THE POSSIBLE HARMS AND SIDE EFFECTS OF PARTICIPATING? Taking a blood sample is felt to have very low risks. The needles used to take blood might be uncomfortable and you may feel lightheadedness, and/or get some minor bruising and/or rarely an infection at the site of the blood draw. 7. WHAT ARE THE BENEFITS OF PARTICIPATING IN THIS STUDY? There may not be direct benefits to you from taking part in this study. We will mail you your blood vitamin D level and suggestions on how to improve vitamin D status, if required. We hope that the information learned from this study can be used in the future to help determine the needs for vitamin D during pregnancy. 8. REIMBURSEMENT In order to defray the costs of transport each participant will receive a grocery voucher. 9. YOUR PARTICIPATION IS VOLUNTARY. Your participation is entirely voluntary, so it is up to you to decide whether or not to take part in this study. Before you decide, it is important for you to understand what the research involves. This consent form will tell you about the study, why the research is being done, what will happen to you during the study and the possible benefits, risks, and discomforts. If you wish to participate, you will be asked to sign this 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. If you do not wish to participate, you do not have to provide any reason for your decision not to participate nor will you lose the benefit of any medical care to which you are entitled or are presently receiving. Please take time to read the following information carefully and to discuss it with your family, friends, and doctor before you decide. 10. WHAT HAPPENS IF I DECIDE TO WITHDRAW MY CONSENT TO PARTICIPATE? Your participation in this research is entirely voluntary. You may withdraw from this study at any time. If you decide to enter the study and to withdraw at any time in the future, there will be no penalty or loss of benefits, if any, to which you are otherwise entitled. The study 145 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. 11. WILL MY TAKING PART IN THIS STUDY BE KEPT CONFIDENTIAL? For the purposes of this study, you will be assigned a study code number, and your name will not be mentioned in any reports or publications of the study results. Your confidentiality will be highly 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 designate by representatives of Health Canada and the UBC Research Ethics Board for the purpose of monitoring the research. All samples and questionnaires will be marked only by code numbers and will not contain any names. No records that identify you by name or initials will be allowed to leave the Investigators’ offices. 12. AFTER THE STUDY IS FINISHED. If you have any questions or desire further information about the study procedures before or during participation, you may contact Dr. Tim Green, at any time, at xxx-xxx-xxxx. 13. WHO DO I CONTACT IF I HAVE ANY QUESTIONS OR CONCERNS ABOUT MY RIGHTS AS A SUBJECT DURING THE STUDY? If you have any concerns about your rights as a research subject andJor your experiences while participating in this study, contact the Research Subject Information Line in the University of British Columbia Office of Research Services at 604-822-8598. 146 14. SUBJECT CONSENT TO PARTICIPATE I have read and understood the subject information and consent form. I have had sufficient time to consider the information provided and to ask for advice if necessary. I have had the opportunity to ask questions and have received satisfactory responses to my questions, and I understand that all of the information collected will be kept confidential and that the results will only be used for scientific objectives. I 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 changing in any way the quality of care that I receive. I am not waiving any of my legal rights as a result of signing this consent form, and I understand that there is no guarantee that this study will provide any benefits to me. I have read this form and I freely consent to participate in this study. I have received a dated and signed copy of this form for my own record. LI I agree that the research team can contact me to consider participating in future studies. I _____________________________ voluntarily give consent to participate in the study (Please print your name) entitled VImitIN D STATUS OF PREGNANT WOMEN IN VANCOUVER Printed name of subject Signature Date Printed name of witness Signature Date Printed name ofprincipal investigator! Signature designated representative Date 147 A pp en di x D :R ec ru itm en tB ro ch ur e In te re st ed in pa rt ic ip at in g? R es ea rc h su pp or te d by H el p u s le ar n m o re a bo ut To R eg is te r C on ta ct : Ti na Li x x x -x x x -x x x x Co -in ve sti ga to rs : M s Ti na Li D rT im G re en D rS he ila In ni s M s Je an ni e D ic ki e M s B ar ba ra Cr oc ke r In pa rtn er sh ip w ith V an co uv er Co as ta lH ea lth St af f BC V , , . (L ’T RC W e re sp ec ty ou rp riv ac y. Y ou ri nf or m at io n w ill re m ai n co n fid en tia l. , ir . V an co uv er - C oa st at H ea lth V ita m in D In Pr eg na nc y 00 V ita m in D is be st kn ow n fo ri ts ro le in st ro ng bo ne s. N ew re se ar ch al so su gg es ts it m ay be im po rta nt in pr ev en tin g so m e di se as es su ch as ca n ce r. W e o bt ai n so m e v ita m in D fro m fo od ;h ow ev er ,m o st o fo u r v ita m in D is m ad e in th e sk in fro m th e su n . Th is is a co n ce rn be ca us e in th e w in te rm o n th st he re is lit tle su n lig ht .V ita m in D is im po rta nt fo re v er yo ne bu ti sp ar tic ul ar ly im po rta nt du rin g pr eg na nc y. Li ttl e is kn ow n ab ou tv ita m in D le ve ls o fp re gn an t B C w o m en . W ho c a n pa rt ic ip at e in th e st ud y? • A ny he al th y pr eg na nt w o m an in he r la te 2 ’ o r ea rly 3rd tr im es te r (ap pro xim ate ly 20 35 w ee ks ) W ha tw ill be e x pe ct ed o fm e? A tte nd an ce at o n e co m m u n ity cl in ic o r o n e v isi t t o BC w o m en ’s ho sp ita l. Th e cl in ic w ill ta ke 30 -6 0 m m . V iL ar iii i I, W ha t w ill ha pp en a t th e cl in ic ? Su rv ey • B lo od sa m pl e • Sk in co lo ur m ea su re m en t W ill I re ce iv e m y re su lts ? Y ou w ill re ce iv e yo ur v ita m in D le ve l, v ita m in D an d ca lc iu m in ta ke . Y ou w ill re ce iv e a gr oc er y v o u c he rt o co m pe ns at e yo u fo r yo ur tr av el ex pe ns es . V ita m in D in Pr eg na nc y A re Pr eg na nt W om en G et tin g E no ug h V ita m in B ? Appendix E: Recruitment Poster Pregnant Women Needed!!! Vitamin D Status of Pregnant Women in Vancouver Are pregnant women in Vancouver getting enough vitamin D? Would you like to help us learn more about vitamin D in pregnancy? Who can participate in the study? Any healthy pregnant woman (w/o pregnancy complications: diabetes, cardiac diseases, kidney diseases, renal diseases, communicable diseases). What wifi be expected of me? Attendance at one community clinic or one visit to BC Women’s Hospital (30-40 minutes). What wifi happen at the clinic? - Survey - Blood sample - Skin colour measurement You will know your vitamin D level. You will also receive a grocery voucher to compensate you for your travel expenses. If you are interested in participating, please contact Tina Li at xxx-xxx-xxxx. Research supported by 4’. flC WOM.PS OSPILi *AL11 IUBc 4 Vancouver CoastatHeatth 150 Appendix F: Demographic and Lifestyle Questionnaire Vitamin B during Pregnancy Study Questionnaire ID { } Please note that all information on this questionnaire is labelled with an ID number only, and stored separately from your name and contact details. All information collected will be stored securely, and personally identfIable information will be accessible to no one except Dr Sheila Innis and Dr Tim Green. SUN EXPOSURE INFORMATION Since we make most of our vitamin D from the action of sunlight on skin the next few questions focus on how much sunlight exposure you receive. The following questions refer to the last TWO MONTHS. 1. In the past TWO MONTHS, how often did you cover up or wear clothing that covered each of the following body parts when you were outside? Head (hat or scarves) 1 Never 2 Sometimes 3 Often 4 Frequently 5 Always Face 1 Never 2 Sometimes 3 Often 4 J Frequently 5 Always Hands 1 Never 2 Sometimes 3 Often 4 Frequently 5 LI Always Le s (pantyhose, long dresses, trousers) 1 Never 2 Sometimes 3 Often 4JJ Frequently 5 f Always Arms (long sleeves) 1 Never 2 Sometimes 3 Often 4 Frequently 5 j Always 2. A) Over the last two months during your work days are you: Mostly indoors / mixture of in and outdoors / mostly outdoors? (please circle) B) Over the last two months, during your workdays, how long have you spent outdoors, on average, each day? 1 Less than 15 minutes/day 2 15 to 30 minutes/day 151 3 [1 30 minutes to 1 hour/day 4 LI 1 to <2 hours/day 5[] 2to<4hours/day 6 [] 4 or more hours/day C) Do you ever work night shift? YES NO Occasionally / fairly often / most of the time (please circle) 3. A) Over the last two months during your leisure days are you: Mostly indoors / mixture of in and outdoors / mostly outdoors? (please circle) B) Over the last two months, during your leisure days, how long have you spent outdoors, on average, each day? 1 Less than 15 minutes/day 2 [1 15 to 30 minutes/day 3 [] 30 minutes to 1 hour/day 4 LI 1 to <2 hours/day sLI 2to<4hours/day 6 [J 4 or more hours/day 4. Have you traveled outside Canada in the last 2 months? YES [] No [j Where? When? ____________ For how long? ____________ Where? When? For how long? 5. Over the last two months how often did you use sunscreen (or moisturizer, make-up etc. that contains sun protection) when you were out in the sun? (please check only one box) 1 [] Never 2 LI Sometimes 3 LI Most of the time 4 LI Always 152 6. What was the Sun Protection Factor (or SPF) of the sunscreen you usually or mainly used? lease check only one box) Didn’t usually use a sunscreen LI LessthanSPFl5 SPF 15 SPF3O Greater than SPF 30 LI Don’t know 7. Did you ever put sunscreen on some parts of your body that were exposed to the sun, but not on other exposed parts? For example, you put sunscreen on your face but did not put it on your arms or legs.[1 YES LINO Which parts of the body was sunscreen used on? 1 LI Face 2 LI Arms 3 LI Legs Other - Specify _____________________ 8. Have you sunbathed over the last two months to try to get a suntan? (By sunbathe we mean that you stayed out in the sun because you wanted your skin to go browner or more golden in color) LI YES LINO How often? ___ ____ For how long each time? ________________ 9. Have you used a sunbed in the last 3 months? LI NO LI Yes, once every 2 weeks LI Yes, more than twice a week LI Yes, monthly LI Yes, once a week LI Yes, every 2 or 3 months For how long each time? ______________ 10. Have you used spray tanning on your arms in the last 3 months? [I NO LI Yes, once every 2 weeks LI Yes, more than twice a week LI Yes, monthly LI Yes, once a week LI Yes, every 2 or 3 months 153 PERSONAL INFORMATION The nextfew questions are taken mainlyfrom last Canadian Census in 2006. The answers to these questions will allow us to compare the people in our survey to the wider Vancouver population. Remember your information will be kept strictly confidential. 1. Date of Birth (Day/Month/Year) 2. Pregnancy week 3. Height Weight (current) Weight (pre-pregnancy) 4. Estimated pregnancy due date 5. Have you smoked tobacco during pregnancy? YES []No If yes, how many cigarettes per day7 6. In what country were you born? If you were not born in Canada what year did you come to Canada to live7 7. What were the ethnic or cultural origins of your ancestors? An ancestor is usually more distant than a grandparent. For example, Canadian, English, French, Chinese, Italian, German, Scottish, East Indian, fish, Cree, Mi’kmaq (Micmac), Métis, Inuit (Eskimo), Ukrainian, Dutch, Filipino, Polish, Portuguese, Jewish, Greek, Jamaican, Vietnamese, Lebanese, Chilean, Salvadorean, Somali, etc. Specify as many origins as applicable 8. Are you an Aboriginal person, that is, North American Indian, Métis or Inuit (Eskimo)? No, Continue with next question []Yes, North American Indian Yes, Metis Yes, Inuit (Eskimo) 154 9. Are you (check all that apply) V/bite Southeast Asian (e.g., Vietnamese,Cambodian, Malaysian, Laotian, etc.) LI Chinese LI South Asian (e.g., East Indian, Pakistani, Sri Lankan, etc.) LI Black LI Filipino LI Latm American LI Arab LI West Asian (e.g., Iranian, Afghan, etc.) LI Korean LI Japanese Other — Specify 10. How many people live in your household7 11. What is your best estimate of the total income, before taxes and deductions, of all household members from all sources in the past 12 months? $ LI Don’t know LI Don’t want to say 12. What is the highest level of education you have completed? LI No schooling LI Some trade! vocational training LI Some elementary LI Completed trade/vocational training LI Completed elementary LI Some university LI Some high school LI Completed university LI Completed high school 155 Appendix G: Food Frequency Questionnaire FOOD FREQUENCY QUESTIONNAIRE Subject code: ________________ Today’s Date: _________________ Please list nutritional supplements used in the past month, using as much detail.as you can remember: BRAND NAME OF SUPPLEMENT or TYPE AMOUNT TAKEN (e.g., Exact calcium 500 mg + vitamin D) (e.g., 1 tablet eveiy other day) How long ago did you take your last maternal supplement if you take one? When did you last eat? 1. We want to know how often you eat or drink certain foods each month. 2. Think about a typical month not just what you ate this week which might be different. 3. Medium portion sizes are given to help you determine the usual size of the food or drink, and to compare to small and large. 4. If you drink or eat much L (approximately half) than the medium portion size described, then check small. If you drink a large glass of milk every day (approximatelyl.5 times the size ofmedium), then check large. 5. Fill out the form similar to this example: - If you drink a carton of chocolate milk (25OmL) Monday through Friday, then choose M (medium) and show it as 5-6 times per week. Type of Never 1 2-3 1 2 3-4 5-6 1 2+ Medium Your or less per per per per per per per per Food or than 1 Mo Mo Wk Wk Wk Wk Day Day serving S ML Drink month Milk: X lcup X whole, 2%, (8 oz or 250 1% or skim mL) Chocolate 1 cup Milk: x (8 oz or 250 x whole, 2%, mL) 1%orskim — — 156 Type of Never 1 2-3 1 2 3-4 5-6 1 2+ Medium Your Serving or less servin° SizeFood or than 1 Mo Mo Wk Wk Wk Wk Day Day s M L Drink per month Dairy Products Milk: whole, 1 cup 2%, l%orskim (8ozor25O mL) Chocolate Milk 1 cup (8 ozor 250 mL) Soy Milk! 1 cup Beverage: (8 ozor 250 Fortified mL) Soy Drink: 1 cup Plain (not (8 oz or 250 fortified) mL) — — — Other plant 1 cup milks (rice, (8 oz or 250 potato, etc) mL) — Milk in coffee 1 Tablespoon or tea Milk on cereal ‘/2 cup (if not included above) Milk shake 1 cup (8 oz or 250 mL) Milk dessert Y2 cup (one (Ice cream, scoop, 1 pudding) container) — — Yogurt (milk or ½ cup (125 soy) g, 1 — container) — — Soft Cheese 1 Tablespoon (cream cheese) Hard Cheese I cube 2” (parmesan, (2 slices) cheddar, etc) Butter (in any I pat; foods eaten) — — — 1 Teaspoon — — — Margarine I pat; I (In any foods Teaspoon eaten) Seafood and Meat Canned 2 salmon Tablespoons or 1 cup of casserole Canned tuna 2 Tablespoons or 1 cup of casserole Canned 2fish(l/2 sardines — can) — — — Salmon steak 90 g (3 oz) — Other fish: 90 g (3 oz) white Other fish: 90 g (3 oz) oily —— — — — 157 Type of Never 1 2-3 1 2 3-4 5-6 1 2+ Medium Your — or less per per per per per per per per servino ServingFood or than 1 Mo Mo Wk Wk Wk Wk Day Day Size Drink per - month Seafood: e.g. 1 cup meat shrimp, crab — — — Beeforpork 90g(3oz) — — Bacon or 2 slices sausage 2 links — — Others Orange Juice: 1 cup (8 oz, FORTIFIED 250 mL) WITH CALCIUM, VITAMIN D Orange Juice: 1 cup (8 oz, not fortified 250 mL) with calcium, vitamin D White bread, 1 slice, I roll, bun, small roll, Y2 biscuit bagel, bagel Nan, tortilla Dark bread, 1 slice, 1 roll, bagel small roll, ‘/2 bagel Taco chips, 1 cup (28 g) nacho chips — — — Waffle, 1 piece pancake, (4” round) French toast Tofu 1 cube (2”) Macaroni with 1 cup cheese Cream soups 1 cup (250 made with mL) milk Taco or burrito 1 regular made with taco; 1/2 cheese burrito Pizza made 1 slice with cheese Lentils, beans, ‘/2 cup peas — cooked — — —- Eggs: eaten I large egg alone or in other foods Potatoes; ‘/2 cup ( I mashed with scoop) milk + margarine Broccoli, kale, 1 cup raw or greens Y2cup cooked 158 Appendix H: Participant Thank You Letter CHILD go West 28th Avenue. Vancouver & FAMILY British Columbia, Canada V5Z 484 RESFARC[ www.ctnca NST[IUTE Date: Dear We thank you for your participation in our Vitamin D during Pregnancy Study. Your participation in this study has made an invaluable contribution to our research. During the past year, we recruited over 340 pregnant women of a variety of ages and ethnicities. We measured vitamin D (25 hydroxyvitamin D) in your blood, the best indicator of vitamin D status. The overall average vitamin D level was 67 nmol/L. This average is somewhat higher than that reported in other studies. Your vitamin D level was nmoIfL. It is generally agreed that levels less than 25 nmollL should be avoided as this may increase the risk of bone disease. Newer research suggests that 75 nmol/L or higher may be required for optimal health although the exact level required remains controversial. Fortunately only 1% of women had vitamin D levels below 25 nmol/L but over half had vitamin D levels less than 75 nmol/L. You may want to discuss your vitamin D level with your family doctor. We obtain some vitamin D from food (milk and fatty fish), but most of our vitamin D is made in the skin from the sun, when it is available. Unfortunately in Canada there is not enough sunlight in the winter months (Oct — Mar) to make vitamin D. Also people with darker skin or who cover up when outside tend to have lower vitamin D levels. Vitamin D supplements maybe required. Health Canada currently recommends that pregnant women receive 200 IU per day. Other organizations such as the Canadian Pediatric Society recommend considerably more (2000 IU). Over 90% of participants were taking vitamin D containing supplements. Most contained 400 IU. This may explain the higher vitamin D levels in our study compared to others. One important question we forgot to ask was: When did you start taking you multi-vitamin supplement and if you discontinued it when? Please complete the attached form and return it in the stamped addressed envelope. Alternatively you can contact Tina Li at xxx-xxx-xxxx. Finally, based on our findings we were funded to do a vitamin D supplementation trial during pregnancy and lactation. If you or anyone you know is interested in participating please contact us. We will be recruiting for this study over the next 1.5 years. A brochure is enclosed. If you have any question please do not hesitate to contact us. Sincerely, Dr. Tim Green (Principal Investigator) Ms Tina Li (Graduate Student) Food, Nutrition & Health Food, Nutrition & Health The University of British Columbia The University of British Columbia 159 Participant ID: Vitamin Supplement Form 1. When did you start taking your multi-vitamin supplement (i.e. Materna)? I did not take a multi-vitamin supplement. Pre-pregnancy ______ Month 5 ___________ Month 1 Month 6 Month 2 Month 7 Month 3 Month 8 Month 4 Month 9 2. If you started but then stopped taking your multi-vitamin supplement when did you stop? I did not stop taking the supplement. Pre-pregnancy Month 5 Month 1 Month 6 Month 2 Month 7 Month 3 Month 8 Month 4 Month 9 160

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