Vitamin D Dose-Response Study to Establish Dietary Requirements in Infants
Condition: Vitamin D Deficiency
MeSH terms: Vitamin D Deficiency
Study type: Interventional
Phase: Phase 2

Summary: 
      The research team is comprised of an expert nutrition scientist and a pediatrician with
      expertise in endocrinology. Both have world-class experience in assessing bone mass in
      infancy. Together they have designed a study to determine how much dietary vitamin D is
      needed to optimize health in infants. This is important since many infants are born with
      vitamin D deficiency. At present the dosage of vitamin D that is optimal for infant health is
      unclear and recent research suggests that vitamin D status very early in life has long
      lasting effects on bone mass and other health issues. Therefore, in a group of healthy
      infants, this research team will test which dosage of vitamin D is needed to achieve optimal
      vitamin D status. Optimal vitamin D status will be based on growth, biomarkers of vitamin D
      and bone status in blood and also general health. The infants will all be breastfed and begin
      the study at about 2 weeks of age. At 3 months intervals over the first year of life, each
      infant will be measured for growth, duration of breastfeeding and supplement use plus have a
      bone density scan to determine changes in bone growth. The data will be helpful to guide
      health care professionals in providing the best care possible for their infants. It will also
      be important to the health of populations since dietary recommendations for vitamin D are
      used to guide fortification of foods and development of evidence based policy around nutrient
      recommendations and their implementation.
    

Detailed description: 
      Vitamin D status is currently judged by serum 25(OH)D and the parathyroid hormone
      (PTH)-25(OH)D dynamic (1). The ultimate effect of vitamin D on human health is a healthy
      skeleton, which is characterized in infancy by normal linear growth and bone mineral
      accretion and absence of bone related disease, such as rickets (1). The importance of these
      indicators is illustrated in epidemiological research, where linear growth in infancy is
      positively related to peak bone mass as an adult (2). Moreover, both vitamin D status (3) and
      intake (4) in infancy are related to bone mass in children.

      In 2004, Health Canada reaffirmed its population health policy that all breastfed, healthy
      term infants in Canada receive a daily vitamin D3 supplement of 400 IU (5). This policy
      recommendation was made in consultation with the Canadian Expert Advisory Committee on
      Dietary Reference Intakes (DRI). They determined that the 1997 DRI value for vitamin D of 200
      IU was too low for Canadians based on Canadian studies (6, 7) and surveillance systems (1)
      that indicated a high rate of vitamin D deficiency and vitamin D deficient rickets continued
      in Canada.

      Both the Health Canada committee (5) and Institute of Medicine Committee on Dietary
      Recommended Intakes (1) acknowledged that there was considerable uncertainty regarding what
      defines optimal vitamin D intake in infancy based on functional outcomes.

      In adults, a minimum serum 25(OH)D value of 75 nmol/L now defines optimal vitamin D status
      (8). This concentration is derived from dose-response studies of the relationship between
      25(OH)D and PTH, where PTH plateaus in approximately the mid-normal range and also from
      studies where 25(OH)D concentrations between 90 and 100 nmol/L are positively related to bone
      mineral density (BMD) in young adults (9). A dose-response study to determine the
      physiological and functional response to vitamin D3 supplementation has not been conducted in
      infants and is necessary to confidently set DRI values for Canadian infants.

      In summary, health care professionals are faced with major gaps in knowledge of: a) what
      constitutes optimal vitamin D status in infancy based on the physiological and functional
      outcomes, 25(OH)D, PTH and bone mass; and b) what oral dosage of vitamin D supplement is
      required to elevate serum 25(OH)D to optimal values as the key biomarker of optimal vitamin D
      status.

      The global objective of this study is to conduct a dose-response study of vitamin D3 intakes
      and to provide information regarding the physiological and functional response to vitamin D
      supplementation in infancy, including: 25(OH)D concentrations and the relationship between
      25(OH)D, PTH and bone mineral accretion. Once the best dosage is determined, funding for a
      larger study spanning all Canadian regions will be sought to more conclusively demonstrate
      that this dosage is appropriate for all Canadian infants. In the larger study, infants will
      be followed longitudinally to confirm that optimization of vitamin D status in infancy
      enhances peak bone mass.

      2.0 BACKGROUND 2.1 Recommendations for Intakes of Vitamin D for Infants The AI value of at
      least 200 IU/d was derived from the amount of breast milk typically consumed by infants and
      the concentration of vitamin D in breast milk that resulted in serum 25(OH)D concentrations
      above 27.5 nmol/L (1). The current recommendations for vitamin D intake in Canada for infants
      are listed below, but no dosage has been evaluated using dose-response studies conducted in
      Canada.

        -  400 IU vitamin D3/d for infants recommended in 2004 by Health Canada, The Canadian
           Pediatric Society and Dietitians of Canada in Guidelines for Healthy Term Infants (5);
           and

        -  that entirely breast-fed infants should be given 400 IU of vitamin D daily that may be
           increased to 800 IU/d from October through April as recommended by the Indian and Inuit
           Committee of the Canadian Pediatric Society (10). Infants bottle-fed formulas made from
           fortified whole or canned milk should receive additional 400 IU from October through
           April (10).

      2.2 Vitamin D Metabolism and Sources of Vitamin D for Infants: The Canadian Scenario
      Endogenously synthesized vitamin D is in the form of cholecalciferol (vitamin D3). Most food
      sources and supplements in Canada have this mammalian form, but some contain the plant form,
      ergocalciferol (vitamin D2). Vitamin D3 is estimated to be approximately 9.5 times more
      potent than vitamin D2 (12).

      Endogenous synthesis accounts for the majority of vitamin D in adults (8). Above latitude 42
      in the northern hemisphere, synthesis is limited to late spring through mid-autumn, because
      of the low intensity of UVB radiation, during the rest of the year. The majority of Canadians
      reside north of the 42nd parallel, placing them at risk for hypovitaminosis D year round
      because of the use of sunscreen and environmental factors, such as clothing and pollution,
      which reduce exposure to UVB sunlight (14). This means that endogenous synthesis in pregnant
      women and subsequent maternal-fetal transfer may be compromised. Fortified foods such as
      milk, margarine and vitamin supplements remain the major source of vitamin D for many
      Canadian adults.

      2.2.1 Maternal-fetal transfer by birth: Given the fact that Canadian women do not consume
      enough milk (15, 16), it is not surprising that over 1/3 of infants are born already
      deficient in vitamin D. In Manitoba, the PI has documented that 36% of infants from white or
      non-white parents are deficient in vitamin D at birth, defined as a serum 25(OH)D below 27.5
      nmol/L, with 46% of their mothers themselves having values < 37.5 nmol/L (6). In that study,
      20% of white infants were deficient, and as a group 60% of First Nations, Asian, Filipino and
      Black infants were deficient. This suggests that vitamin D deficiency is not uncommon,
      particularly in non-white women and their infants (6, 19-22). Likewise season of birth (19)
      and ethnicity (6) are important factors to consider in defining how much vitamin D is
      required to achieve and maintain target values for 25(OH)D in infants.

      2.2.2 Infants from birth to 12 months: Infants have limited stores of vitamin D at birth
      because the amount transferred from the mother has a very short half life of 10-21 days (1).
      For infants exclusively breastfed, vitamin D supplementation is recommended by Health Canada
      (5) because vitamin D concentrations are low in the breast milk of Canadian mothers (4-40
      IU/L). Fortified formula would provide ~ 400 IU vitamin D/L daily, but likely not achieved
      until close to 6 months of age (i.e., with intake 1L/d). After 6 months of age, the foods
      available to infants have limited vitamin D content.

      2.3 Role of Early Life Vitamin D in Human Bone Health Term infants with 25(OH)D
      concentrations above 27.5 nmol/L have higher weight adjusted bone mass than those born with
      lower concentrations (6). The effects of higher maternal-fetal transfer of vitamin D are
      sustained long into childhood, as illustrated by a recent report from Southampton, UK (50N
      latitude): maternal vitamin D status in pregnancy, synonymous with fetal exposure to vitamin
      D in utero, was positively correlated with bone mass in the children measured as late as 9
      years of age (3).

      The effect of various dosages of vitamin D on whole body/skeletal bone mineral accretion in
      the first year of life has not been reported. In the first year of life, it is expected that
      whole body bone mineral content (BMC) will almost triple in parallel to growth (24, 25). In
      formula fed infants, whole body BMC at 1, 3, 6, 9 and 12 months of age are 79, 130, 160, 200
      and 235 g respectively as measured with dual energy x-ray absorptiometry (DXA) with a
      precision error of 4.5% (25). Since the largest increment is between 1 and 3 months (63%),
      this is an ideal time to compare outcomes with respect to effects of various dosages of
      vitamin D on bone mineral accretion. However, this has yet to be conducted and is an
      important objective of the present proposal. It is anticipated that optimal vitamin D status
      will yield greater bone mass, since in Geneva, Switzerland (46N latitude), even a 400 IU/d
      vitamin D3 supplement in infancy (median duration of 12 months) is associated with higher BMD
      at 7 to 9 years of age (4). In the girls who received a vitamin D supplement in infancy, BMD
      was 6% higher in the distal radius and 9% higher in the femoral neck, even after adjustment
      for size (4). These positive associations must be viewed in conjunction with studies
      identifying peak bone mass as a critical factor in bone health (26). Whether the positive
      effects of the supplement translate into higher peak bone mass, is not yet known. An
      additional limitation is that no such studies have been conducted in Canada, with its unique
      climate, culture, food supply, and policies related to infant sun exposure. Since optimal
      vitamin D requirements for Canadian infants are ill defined, the benefits to bone mass are
      also unclear. As such, recommended dietary intake of vitamin D3 for Canadian infants must be
      identified by rigorous, well-controlled, randomized designs with optimal vitamin D status as
      the primary outcome.

      2.4 New Paradigm for Optimal Vitamin D Status Physiologically, when blood calcium falls, PTH
      is released to promote hydroxylation of 25(OH)D to 1,25(OH)2D. Both PTH and 1,25(OH)2D
      mobilize of calcium (Ca) from bone and enhance absorption of Ca from the diet and glomerular
      filtrate. Elevated PTH levels characteristically seen in individuals with inadequate vitamin
      D status can result in the long latency disease of osteoporosis (27). A high PTH can also
      result from low dietary Ca, but for infants, breast milk and formula provide ample Ca.

      Optimal 25(OH)D concentration for infants is unknown but is projected to be at least 75
      nmol/L based on adult studies (8). This value is derived from well controlled dose-response
      studies of vitamin D3 supplementation in men and women, which demonstrate a reduction in
      serum PTH (8, 28, 29) with increasing 25(OH)D concentrations and a plateau in the mid-normal
      PTH reference range. However, current thinking suggests that the 25(OH)D-PTH dynamic is not
      the only instrument to determine recommendations, but should be supplemented with alternate
      biomarkers, such as bone mass and mineral metabolism, to more clearly define what is optimal
      (27). This is illustrated by a study of young adults in whom higher vitamin D concentrations
      (90-100 nmol/L) were associated with higher BMD when compared to those with lower
      concentrations (9). This would suggest that gains in 25(OH)D beyond those associated with the
      PTH plateau are important. Similarly, 25(OH)D concentration of 75 to 80 nmol/L in children 7
      to 18 years of age are related to normalization of PTH (30-34) and has been selected as the
      target concentration for this age group as well. Concentrations of 25(OH)D at and above this
      value are associated with enhanced bone mineral accretion in one cohort (35) and in a
      randomized controlled trial (RCT) of vitamin D supplementation with 400 and 600 IU/d in
      adolescents (36). These studies again emphasize that the PTH-25(OH)D dynamic and bone mass
      are important determinants for establishing the RDA in pediatrics. Such dose-response studies
      of vitamin D3 do not exist for Canadian infants and it is possible that PTH should not be the
      only biomarker of adequacy during this life stage. There are limited dose-response studies
      [2] or RCT [1] examining daily oral vitamin D intake in infants (37-39). The details of these
      studies have recently been summarized as part of a NIH requested systematic review compiled
      by Canadian experts. The key message of this report is that there are no dose-response
      studies of vitamin D3 intake in infants, but only of vitamin D2 (9.5 x lower potency (12)) in
      dosages ranging from 100 to 1000 IU/d. These studies were of short duration and none were
      conducted in Canada. Additionally, not all studies measured PTH and none measured whole body
      bone mass. Clearly, a trial of vitamin D3 in Canadian infants is overdue.

      In order to better understand how to optimize vitamin D status in infants, newer biomarkers
      of bone formation and resorption should be included to more comprehensively assess bone
      metabolism in association with 25(OH)D, PTH and whole body BMC. Suggested biomarkers include
      plasma osteocalcin and urinary N-telopeptide. Osteocalcin increases with growth and bone
      mineralization in the first year of life, as does alkaline phosphatase (40). Osteocalcin is
      the superior marker, since it also reflects seasonal changes in 25(OH)D and PTH in infancy
      (41). For bone resorption, urinary N-telopeptide is a practical marker, as it can be measured
      in urine and is also a specific index for bone resorption. Excretion of N-telopeptide in a
      spot urine sample in infants over the first year of life can be used with confidence for
      comparing groups of patients (42).

      2.5 Safety Considerations, High Dosages Elevate 25(OH)D with No Adverse Effects There are no
      observed adverse effects at vitamin D2 or D3 doses of 1800 IU/d in infants (1). In the event
      of hypervitaminosis D, hypercalciuria and or hypercalcemia could occur. Sustained
      hypercalcemia could manifest as failure-to thrive, anorexia, irritability, nephrocalcinosis
      and its effects, and the remote possibility of cardiac arrhythmias. In one published study
      where infants received approximately 1200 IU of vitamin D per day (D2 supplements and D3
      formula) (43), and in a pilot coordinated Dr. Taback (Univ. Manitoba) and the applicants (see
      CV module, CDA grant) where infants received 2000 IU/d of vitamin D3, no hypercalcemia or
      hypercalciuria was noted. In the later trial, PTH data are not yet available and this dose of
      vitamin D3 results in 25(OH)D well above 75 to 80 nmol/L (199 nmol/L, n=4). While too small
      to calculate SD, this study suggests that the required intake of vitamin D3 is likely below
      2000 IU/D.

      2.6 Theoretical Calculation of Vitamin D Intakes To Establish Study Dosage Groups As
      described in 2.1, the RDA is defined as the EAR + 2 SD and covers requirements of 97% of the
      population. If the target is 75 nmol/L for all infants, this means that the 2.5 percentile
      must be at this concentration. Using data from the PI's research of 83 cord blood samples,
      the mean value of 25(OH)D was 35 with SD of 15 nmol/L (full dataset from Weiler et al. (6)).
      Thus the lower 2.5 percentile is 5 nmol/L. To elevate this to 75 nmol/L, a gain in 70 nmol/L
      would be required (97 % of infants above target). Unfortunately, there is little available
      data as to the dose-response dynamics of mammalian cholecalciferol (D3) in infants, and
      studies of plant ergocalciferol (D2) may be misleading due to differences in bioavailability,
      which may be as much as a whole order of magnitude. The preliminary data from Drs. Taback and
      Weiler using daily supplements of either 400 IU or 2000 IU of D3 in a small number of
      Winnipeg infants (n=4 in each group) are the only data for us to base our estimates. Although
      too small a sample to confidently estimate measurement variance, they observed a rise of 44
      and 115 nmol/L in the respective mean 25(OH)D concentrations in the first 3 months of
      supplementation. Thus, the 400 IU dose of vitamin D3 is expected to achieve a mean
      concentration of 75 nmol/L by 3 months of age. 800 IU D3 will be used in this study because
      this is currently recommended for Canadian infants. Because 2000 IU D3 was felt to be
      excessive based on the modest numbers of infants given this dose and their short follow up,
      we elected to select 1200 and 1600 IU as intermediate yet likely candidate RDA doses.

      3.0 OBJECTIVES and HYPOTHESIS HYPOTHESIS: Daily oral dosage of 400 IU/d of vitamin D3 will be
      inadequate to achieve optimal vitamin D status in 97% of Canadian infants.

      PRIMARY OBJECTIVE: To define the dosage of vitamin D3 required to achieve 25(OH)D
      concentrations ≥ 75 nmol/L at 3 months of age in exclusively breast fed infants.

      SECONDARY OBJECTIVES: When multiple doses achieve our primary objective of optimal 25(OH)D
      concentrations, the best dosage will be identified based on: (i) 25(OH)D-PTH dynamics (ii)
      incidence of adverse events, such as disturbed mineral homeostasis (e.g. hypercalcemia,
      hypercalciuria, etc), and (iii) alternate markers of bone health (e.g. bone mass, osteocalcin
      and N-telopeptide) TERTIARY OBJECTIVE: We will examine the quantitative relationships between
      primary and secondary outcome measures and the various prospectively collected predictor
      variables, including dose, vitamin D from breast milk or fortified formulae, gender, season
      of birth, ethnicity, baseline vitamin D stores and determine if the selected dosage maintains
      25(OH)D over the first year of life.

      4.0 METHODOLOGY: Dose Response Study 4.1 Population n=192 breast-fed infants from greater
      Montreal region will be studied. The rationale for studying breast-fed infants is that this
      is the current recommended optimal food for infants, along with vitamin D supplements, and
      the fact that 85% of women in Canada initially breastfeed their infant (44, 45). Beginning
      with breastfeeding allows confirmation that the best dose is suitable while breastfeeding and
      if this dose is also optimal later in infancy with introduction of foods and weaning to
      vitamin D fortified formula. Initially a pilot study will be employed with 24 infants to
      evaluate the recruitment strategy, study questionnaires and procedures that will be used in
      the larger study.

      4.2 Study Protocol 4.2.1 Recruitment: Rolling recruitment over 1 year with 1-year follow up
      for all beginning in September of 2006 and continuing through August 2007. Equal numbers
      across all months will be sought. At least two large pediatric clinics (Médicentre St-Lazare
      and Clinique de santé jeunesse) will be the primary recruitment centres with over 1500
      newborns annually receive their ongoing medical care. These clinics provide care to families
      with a wide range of SES and cultures representative of Montreal and Canada. Infants will be
      recruited at the first postnatal visit at 2 weeks of age. Such recruitment age is suitable
      since breastfeeding will be deemed as established and recovery of birth weight and good
      health are confirmed. The clinician will inform women and families who meet inclusion
      criteria, and only those who are interested in participating in the study will have their
      names and contact address and telephone number forwarded to the study coordinator. All study
      visits will take place at the Mary Emily Clinical Nutrition Research Unit, School of
      Dietetics and Human Nutrition.

      4.2.2 Study treatment groups: After obtaining written consent the infant will be randomized
      to a study group stratified for sex (see page 12) i for randomization process). At
      recruitment (2-4 weeks of age), baseline measurements will be conducted prior to providing
      treatment. Infants will then be randomized to receive 400, 800, 1200 or 1600 IU/d of vitamin
      D3 to 1 year of age. A placebo group is not included since the standard in Canada is 400 IU/d
      and a precedent for considering placebos unethical has already been set (37). The supplement
      will be double blinded to the investigators and all staff by coding of the supplements by
      Euro-Pharm who as agreed to provide the supplement in kind. This supplement will be stable
      for at least 3 months (the longest interval of measurements) and each dosage delivered in 2
      ml volumes.

      4.2.3 Frequency and duration of follow up: Baseline and all follow up visits (3, 6, 9, and 12
      months of life) will include anthropometric measurements and blood and urine analysis for
      (25(OH)D, PTH, calcium, phosphorus, creatinine) as well as bone markers and bone mass
      measurements. An additional blood sample will be obtained 1 month after starting the study to
      monitor blood calcium levels. Bottles of the supplement will be assessed for compliance at
      these visits. Other sources of vitamin D intake will be reviewed by diet history as well as
      determining the content of vitamin D in the breast milk. Maternal baseline information
      regarding demographics will be gathered at the initial visit and dietary vitamin D intake
      will be assessed at each visit.

      4.3 Details of Measurements INFANT: NUTRITIONAL AND HORMONAL STATUS AND BONE METABOLISM 4.3.1
      Sample procurement: Blood and urine samples will be collected in infants at all visits. All
      samples will be taken between 8 and 10 am to control for diurnal variation permitting 3 to 4
      infants to be seen in our Unit daily. Heparinized blood (~1 ml in infants via finger poke -
      400 ul for safety, 600 ul for other measurements) and non-pharmacological approaches used to
      provide for pain control including swaddling and 1 ml 33% sucrose solution given sub lingual
      2 minutes prior to collection (46).

      4.3.2 25(OH)D and PTH concentrations: Vitamin D status will be measured using a RIA (25-50;
      Diasorin) that is known to measure both D2 and D3 in plasma (47). This method (RIA) is
      selected as the now most common clinical method over HPLC and competitive protein binding
      assays. Serum intact PTH will be measured using an ELISA (50 ul; Immutopics International).

      4.3.3 Bone markers: Change in bone metabolism in response to 25(OH)D will be assessed by a
      marker of osteoblast activity related to mineralization, plasma osteocalcin (20 ul;
      Diasorin). Osteoclast activity will be assessed by measuring urinary N-telopeptide (ELISA,
      Osteomark) corrected to creatinine. The assay is specific to Type 1 collagen with a CV% <8%.
      N-telopeptide measurements urine selected over blood to reserve the small blood sample for
      other measurements. Urinary N-telopeptide in spot urine samples is a validated measurement in
      infancy (42). The PI is experienced in all of these measurements (6, 48).

      4.3.4 Anthropometry: For infants, size at birth (weight, length, head circumference) and
      gestational age will be documented from the vaccine carnet. Growth will be assessed each
      visit using triplicate measurements of weight (to the nearest g without clothing/diaper),
      crown-heel length (to the nearest 0.1 cm using an infant length board) and head circumference
      (to the nearest 0.1 cm using a non-stretchable tape). Data will be expressed in absolute
      units and standard deviation scores using the data from the Centers for Disease Control at
      each age. Quality of growth achieved after term age will be available as lean and fat mass
      provided during measurement of bone mass using DXA, reported as accurate and reproducible
      (49).

      4.3.5 Nutrition: Milk intake in infancy will be assessed by weighing infants prior to and
      after breast-feeding (i.e., test weighing and the basis of the required weigh scales). For
      this purpose, parents will be provided with electronic weigh scales designed for test
      weighing over 3 days then returned to the research site by courier. Additionally, breast milk
      vitamin D (all forms) will be measured (using HPLC (51, 52)) in samples collected at
      inception and the 3 month visit using an electronic breast pump. Mothers will be asked to
      feed the infant on one breast and pump from the other. Total intake of vitamin D will then be
      derived from milk intake and milk vitamin D concentration. Thus total intakes of vitamin D
      will be possible whether breast-fed or fed formula.

      At each age, dietary and supplement intake will be documented in infants using written 3-day
      records that are designed to accommodate test weighing and documentation of other milks and
      any foods consumed as successfully used previously by the PI (53). Estimated portions sizes
      will be made using household measuring tools. Mothers will be asked about timing of
      introduction of solid foods or fluids other than breast-milk or vitamin D supplements. If
      infants wean to infant formula or cows milk, the product brand will be documented. It is
      anticipated that by 3 to 4 months of age that many infants will wean from breastfeeding to
      formula. However, the dosage will not be adjusted. Intake of vitamin D from infant formula,
      cows' milk etc will be documented and used to calculate total daily intake above the
      supplement. Such additional vitamin D will be addressed in the statistical analysis and is
      anticipated to be a systematic variation in total intakes.

      4.3.6 Bone mass: Measurement of bone area, BMC and BMD of the whole body, lumbar vertebrae
      (L2-4) and femur at all visits will be conducted using DXA (QDR 4500A Discovery series,
      Hologic Inc.) at the Mary Emily Clinical Nutrition Research Unit. DXA has been validated for
      measuring infant whole body and regional BMC (49, 54, 55) and delivers minimal radiation (6
      uSv) in comparison to standard pediatric x-rays (~60 uSv). Since positioning in infants is
      difficult to standardize, only BMC is used for whole body and femur. For lumbar spine,
      standardized positioning is feasible enabling assessment of bone area, BMC and BMD. Infants
      will not be sedated and will be scanned while sleeping and wrapped in a receiving blanket and
      standardized clothing without any metal zippers, clasps etc. Data will be expressed as
      absolute values for bone area, BMC and BMD and also as a rate of change for BMC. The
      measurement of whole body bone mass is ideal and recommended by ISCD (56). To enable complete
      data regarding bone, the next best measurement is lumbar spine, vertebra 1-4 (56). This
      measurement is easily attainable in infants to 1 year of age and only requires 30 to 60
      seconds compared to whole body at 3 minutes. In addition, the femur can be scanned in 30 to
      60 seconds and offers a novel assessment of a long bone. Whole body BMC will be corrected to
      body weight, length and lean mass and also expressed as change over the period of study.
      Correction to weight or length is endorsed by ISCD rather than more novel methods such as use
      of bone area and height (59) since normative data for infants is not currently available.
      Differences in whole body bone mineral accretion between inception, 3 and 6 months and
      between 6 and 12 months are greater than the error of measurement by DXA estimated at <4.5%
      (25, 49). Values for spine bone area, BMC and BMD and femur BMC will be compared among
      groups.

      MATERNAL CHARACTERISTICS 4.3.7 General demographics and anthropometry include: Maternal age,
      weight gain in pregnancy, height, family income range and number of dependent members,
      employment, highest education level achieved, previous pregnancies, live births. To obtain
      accurate measurement of height, mothers will be measured at the inception visit. Weight will
      also be measured to accurately describe the population. Self-reported weight gain in
      pregnancy will be documented. Mothers will be asked to self-identify ethnicity. These data
      will be used to simply characterize the study population.

      4.3.8 Nutrient intake: Dietary and supplement intake by the mothers during the last trimester
      of pregnancy will be assessed using a validated food frequency questionnaire, the modified
      Willett/Harvard (60). This questionnaire is modified to assess intakes over the last 3 months
      of pregnancy. These data will be used to describe vitamin D exposure of the fetus in utero
      when bone is mineralizing. At each visit and while the mother is lactating, intake of vitamin
      D will be assessed using a 24-hour recall. The value for maternal vitamin D intake will be
      used to describe our population as necessary to determine if results can be extrapolated to
      the Canadian population.

      4.4 Recruitment and Statistical Analysis 4.4.1 Recruitment: Infants will be equally allocated
      to the 4 dosage groups, based on a randomization in blocks of 8 to accommodate stratifying by
      gender since growth diverges in male and female infants by the first year of life. Even
      distribution among the 4 seasons (standard calendar dates) will be sought by recruiting
      1/12th of the sample each month. The fact that some infants may have already began vitamin D
      supplements will be considered as a systematic error.

      4.4.2 Power and sample size: The sample size will be 48 to enable balanced groups across
      seasons and genders. This sample size will permit analysis of the data by feeding type:
      infants exclusively breast-fed versus weaning to formula as necessary to meet objective 3.

      4.4.3 Data Analysis: The intention-to-treat principle will be applied, including all
      randomized infants for each outcome. For comparison purposes, results will also be evaluated
      in terms of the dose actually received. Should baseline imbalances occur between groups
      despite randomization, these will be treated as covariates and adjusted for. To compare
      proportions between treatment groups, sample proportions will be evaluated by a Chi-squared
      test of proportions, adjusted for multiple comparisons to ensure a family-wise error rate of
      0.05. Results will be assessed at the each time point, with a Chi-squared test for trend in
      proportions to compare their temporal evolution. Mean 25(OH) D concentrations, bone mass, and
      biochemical indices of bone remodeling will be compared in each dose group under a fixed
      effects ANOVA model, with significant group differences localized by suitable post-hoc
      testing (e.g. Tukey method), again with adjustment for multiple comparisons to ensure a
      family-wise error rate of 0.05. Time course will be evaluated under a mixed effects ANOVA
      model (with the addition of time as a random effect). The dynamics of the 25(OH)D-PTH
      relationship is visually assessed in linearized log-log plots, whose slope is a measure of
      PTH sensitivity to 25(OH)D. The best-fit line is determined by least squares, and slopes
      compared by standard methods based on the distribution of regression coefficients as
      t-statistics. Taking the following as independent predictor variables - dose, source of
      vitamin D, age, gender, season of birth, ethnicity, and baseline vitamin D stores - their
      effect on the likelihood of achieving our therapeutic target will be evaluated by multiple
      logistic regression, with ordinary multivariate linear regression to examine their impact on
      serum concentration of 25(OH)D, bone mass, osteocalcin, and N-telopeptide.
    

Intervention name(s): Vitamin D; Vitamin D; Vitamin D
Intervention type(s): Drug; Drug; Drug
Intervention description(s): 400 IU vitamin D / day; 800 IU vitamin D / day; 1200 IU vitamin D / day
Intervention MeSH terms: Vitamin D; Ergocalciferols; Cholecalciferol; Vitamins
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: Triple (Participant, Care Provider, Investigator)
Primary purpose: Treatment
Keywords: Infants; Vitamin D; Dose-response