Chapter  158:  Effectiveness and Safety of Vitamin D in Relation to Bone Health

Search Strategy

Using the Ovid interface, we searched the following databases: MEDLINE ® (1966 to June Week 3 2006); Embase (2002 to 2006 Week 25); CINAHL (1982 to June Week 4, 2006); AMED (1985 to June 2006); Biological Abstracts (1990 to February 2005); and The Cochrane Central Register of Controlled Trials (CENTRAL; 2nd Quarter 2006). No language restrictions were applied, and studies were restricted to human subjects.

Eligibility Criteria

Studies for inclusion were limited, wherever possible, to randomized, controlled trials (RCTs) in order to minimize bias. Inclusion criteria for question one were broadened to include prospective cohorts, case-control and before-after studies due to the lack of studies addressing the association between serum 25(OH)D concentrations and bone health outcomes, especially in infants and children. Question four was restricted to existing systematic reviews to limit scope.

Studies that assessed vitamin D₂ or D₃ with or without calcium supplementation were included. We did not include randomized trials that used calcium with vitamin D as a control arm unless a placebo or lower dose vitamin D arm was also available as a comparator due to difficulty interpreting cause and effect. Vitamin D preparations, calcitriol or alphacalcidol, were not included since they are not considered nutritional supplements and have a different safety profile than vitamin D₂ or vitamin D₃. Studies evaluating the efficacy of vitamin D for the treatment of secondary causes of osteoporosis (e.g., glucocorticoid-induced osteoporosis, renal or liver disease) or for treatment of vitamin D-dependent rickets were not included, in an effort to minimize clinical heterogeneity and since non-dietary sources of treatment are often used as the primary treatment for some of these conditions.

Study Selection

The results of the search were assessed using a three-step process. First, bibliographic records, including title, keywords and abstract, were screened by one reviewer. Potentially relevant records were then screened independently by two reviewers using the full text report and strict eligibility criteria. Conflicts were discussed and resolved through consensus or adjudication by a third reviewer, if needed. Relevant studies were subsequently assessed for study design and categorized by question. The reasons for exclusion were noted using a modified QUOROM format.

Data Extraction

Two reviewers abstracted data on study and population characteristics, type of 25(OH)D assay, vitamin D intervention (type, dose, frequency), co-interventions, reported confounders or covariates and relevant bone health outcomes. One reviewer completed the primary extraction that was then verified for completeness and accuracy by a second reviewer. Differences were resolved through consensus or adjudication. Evidence tables were constructed for each of the included studies, and summary tables were prepared in order to maximize consistency in identifying pertinent data for synthesis.

Assessment of Study Quality

An experienced reviewer assessed the quality of reporting. For the RCTs, the study quality was evaluated using the validated Jadad scale. A Jadad score of ≥ 3 (out of a possible 5) was used to indicate studies of higher quality. Allocation concealment was assessed as adequate, inadequate or unclear. For the observational studies, the methodological quality (poor, fair, good) was evaluated using the grading system adapted from Harris and colleagues.

For each section, an aggregate level of evidence (good, fair, inconsistent) was rated based on quantity, quality and consistency of results. Good evidence (e.g., for or against an association between serum 25(OH)D concentrations and a bone health outcome) was determined by consistent results across studies and at least one study of good quality. Fair evidence was evidence sufficient to determine an association but was limited by consistency of results, quantity, or quality (i.e., no studies graded as good). Inconsistent evidence was defined by an inability to make a conclusion for or against an association, in that studies had conflicting results.

Data Synthesis

Where possible, meta-analysis of RCTs that assessed interventions, populations and outcomes (e.g., fractures or falls) was conducted using a random effects model, with an assessment of statistical heterogeneity. For continuous outcomes (e.g., serum 25(OH)D concentrations, and BMD), the difference in means between treatment groups was used for the meta-analyses. The absolute change in 25(OH)D concentrations was used for quantitative pooling. A weighted average method was used to calculate the 25(OH)D values for the combined treatment group and placebo group. The difference in means was then calculated using the weighted averages for the two combined groups. For dichotomous outcomes such as falls or fractures, RCTs were grouped by type of vitamin D supplementation and whether calcium was used as a co-intervention since we expected there might be different treatment effects with vitamin D₂ versus D₃, and to try to separate out the differential effects of calcium and vitamin D intake. These groupings were then used to minimize clinical heterogeneity in pooled estimates. Summary odds ratios were calculated using the number of individuals who had an event (e.g., fracture). To avoid differences in the reporting of units for 25(OH)D concentrations (i.e., nmol/L, ng/mL, μg/dL, μg/L), all values were converted to nmol/L that was the unit used for data synthesis.

Role of Vitamin D in Bone Health

The principal physiologic role of vitamin D is to maintain calcium homeostasis although it also has potential non-calcemic actions.¹¹ Its principal sites of hormonal action are the intestine, where it increases calcium absorption, and bone. Vitamin D ensures the mineralization of the organic matrix of bone, and also mediates the release of calcium and phosphate from bone to achieve mineral homeostasis.

In the classical endocrine pathway, vitamin D enters the circulation attached to a D-binding protein, is first hydroxylated in the liver to 25(OH)D and then in the kidney to form the active metabolite, 1,25 dihydroxyvitamin D (1,25-(OH)₂ D) or calcitriol. The production of calcitriol is stimulated by parathyroid hormone (PTH), and decreased by calcium.¹² Calcitriol also downregulates its own production. 1,25-(OH)₂D exerts its effects through the vitamin D receptor leading to gene expression and by more immediate effects mediated by second messengers.¹² Calcitriol and the vitamin D receptor are essential for active calcium absorption from the gut, longitudinal bone growth and the activity of osteoblasts (cells that form bone) and osteoclasts (cells that resorb bone). In osteoblasts, vitamin D receptor activation induces expression of the ligand RANKL (receptor activator of nuclear factor kappa beta ligand) on their surface membrane. Interaction of RANKL with its receptor, RANK, on preosteoclasts induces differentiation and activation of osteoclasts.¹³

The enzyme that catalyzes the synthesis of 1,25-(OH)₂D in the kidney, 25-hydroxyvitamin D₃-1-α-hydroxylase, is also expressed in other tissues and cells such as colon, prostate, mammary gland, macrophages, antigen-presenting cells, osteoblasts and keratinocytes, resulting in extrarenal production of calcitriol.⁵ Vitamin D receptor (VDR) expression is also widespread, and many genes encoding proteins involved in the regulation of cell proliferation, differentiation and apoptosis (programmed cell death) contain vitamin D responsive elements. In addition to its calcemic actions, calcitriol has potential immunomodulatory and antiproliferative effects through autocrine and paracrine pathways. These actions have implications for its potential use as a preventive or therapeutic agent in cancer and other chronic conditions, as well as a role in innate immunity.⁵–⁷

Vitamin D promotes active transport of calcium predominantly from the small intestine. At higher calcium loads, more calcium is absorbed by passive absorption, and there is less dependency on vitamin D.

In the vitamin D deficiency state, calcium absorption from the gastrointestinal tract is decreased.¹⁴ A low serum calcium stimulates the production of PTH which regulates calcium homeostasis by increasing the conversion of vitamin D to its active form. This in turn mobilizes calcium from bone, increases intestinal calcium absorption, and decreases calcium excretion.

The decline in estrogen that occurs after menopause is associated with a negative calcium balance, as a result of decreased calcium absorption and increased urinary calcium loss. Menopause is also associated with increased bone turnover, bone loss and reductions in circulating total 1,25-(OH)₂ D concentrations. The effect of menopause on PTH is less clear, with some studies suggesting that estrogen may modulate PTH secretion directly.

The active metabolite of vitamin D is important for the transport of calcium across the placenta in order to provide the fetus with mineral, especially during the last trimester. During pregnancy, fractional calcium absorption increases from 35 (non-pregnant state) to 60 percent during the third trimester. Serum 1,25-(OH)₂D concentrations increase to facilitate the increased calcium absorption although the underlying mechanism is not fully understood, and increased serum PTH concentrations have not been demonstrated.¹⁵

During lactation, neither 1,25-(OH)₂D serum concentrations nor calcium absorption are increased. The usual daily loss of calcium ranges from 280–400 mg and in order to meet these demands, skeletal calcium is released by temporary bone demineralization. Bone demineralization is reversible following weaning.^{16, 17} The extent to which the adaptive processes in calcium homeostasis that occur during pregnancy and lactation depend on maternal vitamin D status, and how this impacts on the mother's bone health, has not been well studied.

Consequences of Vitamin D Deficiency on Bone Health

Vitamin D deficiency is associated with increased bone remodeling which contributes to structural damage, including increased cortical porosity. During skeletal development and growth, severe vitamin D deficiency results in rickets, a mineralization defect of the growth plate.¹⁸ The manifestations of rickets include growth failure, muscle weakness, fractures and skeletal deformities. Severe degrees of vitamin D deficiency in the adult result in impaired mineralization of new bone (osteoid) and osteomalacia.

Less severe degrees of vitamin D deficiency lead to secondary hyperparathyroidism. PTH secretion is stimulated to maintain serum calcium levels and results in increased bone turnover and bone loss, and may lead to osteoporosis.

There is a growing recognition that milder or subclinical degrees of vitamin D deficiency, termed insufficiency, may also be associated with suboptimal health outcomes. Various definitions for both vitamin D deficiency and insufficiency have been proposed that may depend on the particular 25(OH)D assay used as well as the functional outcome measured. There is no clear consensus on the optimal definitions of either vitamin D deficiency or insufficiency. Recent relatively high prevalence estimates of vitamin D insufficiency in the general population^{19, 20}may be attributed to the use of higher 25(OH)D thresholds to define low vitamin D status, compared to previously used thresholds.

Populations at Risk of Vitamin D Deficiency

Overt vitamin D deficiency in the general population is low.^{21, 22} Although vitamin D deficient or nutritional rickets was thought to have been eliminated, contemporary cases have been reported in the literature. It has not been possible to confirm whether the reported cases represent an actual increase in prevalence of rickets over time or reflect an increased awareness of the disease.^{23, 24} Vitamin D-deficiency rickets has been noted more often in dark skinned infants who are breast-fed by mothers who are not vitamin D replete. Infants who are exclusively breast-fed and those whose primary milk source is human milk are at risk.²⁵–²⁷ However, a preliminary study suggests that infants who are breast-fed by vitamin D replete mothers taking high doses of supplemental vitamin D₃ achieve similar circulating vitamin 25(OH)D levels as those infants receiving oral vitamin D₃.²⁸

Older adults manifest vitamin D insufficiency or deficiency for a variety of reasons, including less efficient skin synthesis of vitamin D₃ and a lack of sunlight exposure.^{10, 29}–³² The prevalence of vitamin D deficiency in cohorts of hip fracture patients has been reported at 50% (serum 25(OH)D ≤ 30 nmol/L)²⁹ and 69% (serum 25(OH)D < 50 nmol/L).^{29, 33, 34} A high prevalence of vitamin D deficiency has also been noted in medical inpatients and shut-in individuals.^{35, 36} Vitamin D deficiency is also more common in adults who cover their skin for cultural reasons and dark skinned individuals.^{10, 29, 33, 34, 36}–³⁹

At latitudes above 42 degrees N, ultraviolet energy is inadequate in winter months for the photoconversion of 7-dehydrocholesterol to previtamin D₃. As a result, even in the general population, the prevalence of vitamin D insufficiency and deficiency increases during the winter months.¹⁹ Large seasonal fluctuations of circulating 25(OH)D concentrations can occur, and summer sun exposure may not sustain adequate vitamin D levels over the winter months in northern latitudes for most individuals.⁴⁰

Definitions of Optimal Vitamin D Status for Bone Health

Serum 25(OH)D, the most abundant circulating precursor of active vitamin D, is the most widely accepted indicator of vitamin D status and reflects combined contributions from cutaneous synthesis, and dietary intake including fortified foods and supplemental sources of vitamin D. Serum 25(OH)D has a half-life of approximately two to three weeks, and varies over a wide range. In contrast, the active form of vitamin D, 1,25-(OH)₂D, has a short circulating half-life and is tightly regulated over a narrow range by parathyroid hormone, calcium and phosphate. Serum 1,25-(OH)₂D is not a good measure of vitamin D status since a decrease may not occur until vitamin D deficiency is severe.

There is considerable debate on how best to define adequate vitamin D status for bone health. Various cutpoints of serum 25(OH)D concentrations have been proposed ranging from 40 to 120 nmol/L. This confusion has arisen from two main sources: differences in the functional endpoint (e.g., fractures, serum PTH) and differences in the analytical methods to measure serum 25(OH)D.⁴¹

Endpoints to help define adequate vitamin D status for bone health range from biochemical markers (PTH) and other surrogate markers (e.g., BMD) to clinical endpoints such as fractures. For example, serum 25(OH)D concentrations below 20 to 25 nmol/L have been associated with an increased risk of clinical, radiological and histological changes of osteomalacia and rickets. Concentrations above which bone loss is minimized and fracture risk decreased are other endpoints that have been used. Bischoff found the association between serum 25(OH)D concentrations and BMD had a steep positive slope in the reference range, reaching a plateau at a concentration of 90 to 100 nmol/L in an older Caucasian population.⁴²

As serum 25(OH)D levels increase, serum PTH falls and then levels off. The threshold concentration of 25(OH)D above which there is no further suppression of PTH has also been used to distinguish adequate vitamin D status from vitamin D insufficiency.^{41, 43, 44}

Another outcome that might exhibit threshold behavior is intestinal calcium absorption. Heaney reported that postmenopausal women (mean age 64 years and BMI 28.8 kg/m²) with serum 25(OH)D concentrations at the low end of the reference range may not be maximizing their calcium absorption.³⁹ However, another study did not find evidence for a threshold of 25(OH)D in association with calcium absorption.⁴⁵

Measurement of Serum 25(OH)D Concentrations

There are a variety of assays that measure 25(OH)D. Technical challenges in determining an individual's true circulating 25(OH)D level include the protein's hydrophobic and hydrophilic properties and the strength of its binding to vitamin D binding protein. The available assays vary in complexity of sample preparation, the technical expertise required to run the assays, degree of automation and ability for high throughput, and accuracy of detection of total 25(OH)D and individual isoforms as well as other vitamin D metabolites. Assays include competitive protein binding assays (CPBA), radioimmunoassays (RIA), enzyme-linked immunoassays (ELISA), chemiluminescence assays, high performance liquid chromatography (HPLC) with UV detection, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), and gas chromatography coupled with mass spectrometry (GC-MS).⁴⁶–⁴⁸

The first competitive protein-binding assays (CPBA) required an extraction step, chromatographic clean up for co-extracted contaminants, and radioligand detection. The most recent generation of CPBA includes a chemiluminescent assay that utilizes a specific antibody as a competitive binder and does not require sample extraction. With the availability of commercial assays (e.g., immunoassays), large numbers of samples can be processed rapidly. Chromatographic techniques (HPLC, LC-MS/MS, GC-MS) have the advantage of measuring 25(OH)D₂ and 25(OH)D₃ separately. The highest attainable analytical accuracy may be through the principle of isotope dilution with stable isotope-labeled internal standard compounds and mass spectrometry.^{48, 49}

It is apparent that results differ between methods, and that different methods may not recognize 25(OH)D₂ and 25(OH)D₃ equally. Data from the international Vitamin D External Quality Assessment Scheme (DEQAS) provide an indication of the relative performance of participating laboratories.^{50, 51} DEQAS and other comparative studies have shown that results can differ between laboratories even using the same method.^{50, 52}–⁵⁸ Some studies have reported discordant results in terms of the variability observed, and some have not included an accurate reference method (e.g., LC-MS/MS or HPLC). Even within a particular technique such as RIA, different sources of antibodies may vary in specificity and the ability to detect each isoform, and extraction or purification procedures may also differ.⁵⁴ Some RIA assays underestimate 25(OH)D₂. There are no commercially available standard reference preparations or calibrating materials to help reduce the variability of results between methods and/or laboratories, or to alert the laboratory of any deviation from the true value. Until we can reliably measure serum 25(OH)D concentrations, this important issue must be considered when defining a cut-off point for adequate vitamin D status. It is also possible that assay-specific decision limits may be required in order to define appropriate thresholds, providing further challenges in implementation of recommendations.⁵⁹

Vitamin D Supplementation

Vitamin D₃ (cholecalciferol) is a naturally occurring form of vitamin D. Vitamin D₂ (ergocalciferol) is found in some plants, dietary supplements, and multivitamins. Both forms of vitamin D are biologically inert and must undergo hydroxylation in the liver and kidneys (or in extra-renal sites) to produce the active metabolite. The average increment in serum 25(OH)D concentrations has been estimated at 1–2 nmol/L for every 40 IU (1 microgram) of vitamin D₃ given as an oral dose, depending on baseline 25(OH)D concentrations. Heaney demonstrated that in young men (mean age 38.7 years and BMI 26.2 (2.4) kg/m²), 40 IU of vitamin D₃ (1 microgram) resulted in an increment of 0.7 nmol/L or less when given to individuals with higher baseline levels of 70 nmol/L.⁶⁰ Some studies have reported that vitamin D₂ supplements is less effective than vitamin D₃ (cholecalciferol) in terms of the effect on serum 25(OH)D concentrations, suggesting that vitamin D₂ and D₃ may be utilized differently by humans.⁶¹ The two isoforms may be metabolized differently, and vitamin D₂ has diminished binding to vitamin D binding proteins in plasma.⁶²

Current Dietary Reference Intakes for Vitamin D

The 1997 Institute of Medicine (IOM) Committee was unable to establish estimated average requirements (EAR) on which to base recommended daily allowances (RDA) for vitamin D. The various sources that contribute to circulating 25(OH)D concentrations make this a challenge, and insufficient studies were available to define an RDA. Adequate intakes (AI) were provided instead. Adequate intakes are the amount needed to maintain a defined nutritional state or criterion of adequacy e.g., prevention of rickets or osteomalacia, in all members of a specific healthy population.⁴ Current dietary reference values are based on vitamin D intakes associated with total circulating 25(OH)D levels. The Institute of Medicine's adequate reference intakes for vitamin D are: 200 IU (5 μg/day) for children from infancy to 19 years; 200 IU (5 μg/day) for individuals aged 19 to 50 years; 400 IU/day (10μg/day) for adults 51 to 70 years of age; and 600 IU/day for adults over the age of 70.^{4, 63} The AIs for children up to 19 years and adults aged 19 to 50 years represent decreases from prior reference intakes of 400 IU.^{4, 63} The AI for infants is based primarily on data from the United States, Norway and China that showed a vitamin D intake ≥ 200 IU will prevent physical signs of vitamin D deficiency and maintain serum 25(OH)D above 27.5 nmol/L.⁶³ Vitamin D intakes required for optimal bone health are less well-defined for children and adult populations, especially for those living at northern latitudes.

The more recently published 2005 Dietary Guidelines for Americans (for individuals two years of age or older) recommend higher daily dietary vitamin D intakes (1,000 IU) for individuals who are exposed to insufficient UV-B light, older adults, and people with dark skin.⁶⁴ Concerns about toxicity include the potential for high vitamin D intakes to cause hypercalcemia, hypercalciuria, renal stones and soft tissue calcification. The current tolerable upper limit of vitamin D intake for infants is 1,000 IU and for children and adults, including pregnant and lactating women, is 2,000 IU.⁴

Search Strategy

An initial search for systematic reviews related to vitamin D was conducted, and the review team and Technical Expert Panel (TEP) identified reviews relevant to each of the five research questions. These aided in the development of the search strategy for primary studies. Conceptual analysis was undertaken by one information specialist, and translation of the concepts and the Boolean logic of their combinations were confirmed by a second information specialist. No language restrictions were applied. Using the Ovid interface, the following databases were searched: MEDLINE ® (1966 to June Week 3 2006); Embase (2002 to 2006 Week 25); CINAHL (1982 to June Week 4, 2006); AMED (1985 to June 2006); Biological Abstracts (1990 to February 2005); and The Cochrane Central Register of Controlled Trials (CENTRAL; 2nd Quarter 2006). The MEDLINE ® search strategy is in Appendix A ^b. Adjustments were made to the search when run in other databases to account for differences in indexing. All records were downloaded and imported into the Reference Manager software, and duplicate records were removed. This review underwent a formal update process following completion of a first draft report and prior to final submission with initial searches run in 2005. The dates of the initial search were as follows: MEDLINE ® (1966 to July Week 4 2005); Embase (2002 to 2005 Week 32); CINAHL (1982 to March Week 4, 2005); AMED (1985 to April 2005); Biological Abstracts (1990 to February 2005); and The Cochrane Central Register of Controlled Trials (CENTRAL; 1st Quarter 2005).

Eligibility Criteria

Published English-language studies, examining the safety and/or efficacy of vitamin D in humans, were eligible for inclusion, as follows:

• 1

  The association between serum 25(OH)D concentrations and bone health outcomes was examined in the following populations: 1) children (0 to 18 years); 2) women of reproductive age (19 to 49 years) and; 3) elderly men (≥65 years) and postmenopausal women (50+ years). Bone health outcomes included: BMD, BMC, fractures, falls, performance measures related to falls (e.g., muscle strength or balance) (age group 3 only), calcium absorption (age group 2), calcaneal ultrasound (age group 2), PTH (age groups 1 and 2), rickets (age group 1). Study designs: RCTs, prospective cohorts, before-after and case-control studies.

• 2

  The effect of vitamin D from dietary sources (including fortified foods and/or vitamin D₂ or D₃ supplementation) and sun exposure, on serum 25(OH)D concentrations was examined in the age groups listed above. Vitamin D₂ and D₃ were evaluated separately. Study designs: RCTs of dietary intake/supplementation/sun exposure interventions.

• 3

  The effect of supplemental vitamin D₂ or D₃ alone or in combination with calcium on bone mineral density, fractures, and/or falls was examined in: 1) women of reproductive age (19 to 49 years); 2) postmenopausal women (≥ 50 years) and; 3) elderly men (≥ 65 years). Study designs: RCTs.

• 4

  The relation between sun exposure, serum 25(OH)D concentrations and the risk of non-melanoma and/or melanoma skin cancer was evaluated. Study designs: existing systematic reviews.

• 5

  The potential toxicity of supplemental vitamin D in doses above the adequate reference intakes (e.g., hypercalcemia, nephrolithiasis, soft tissue calcification) was examined in different age groups. Study designs: RCTs.

Systematic and narrative reviews were excluded for all questions except for question 4. However, recent reviews were hand searched for additional potential primary studies that may be pertinent to all questions. Randomized trials of other osteoporosis therapies that included calcium and vitamin D as a control arm were not included unless they also included a placebo or lower dose vitamin D arm that would allow a comparison. Studies evaluating the efficacy of vitamin D for the treatment of secondary causes of osteoporosis (e.g., glucocorticoid-induced osteoporosis, renal and liver disease) or for treatment of vitamin D-dependent rickets were also not considered, in an effort to minimize clinical heterogeneity and since non-dietary sources of treatment are often used as the primary tereatment for some of these conditions. We restricted our inclusion criteria to studies of vitamin D₂ (ergocalciferol) or D₃ (cholecalciferol). Studies that evaluated the efficacy of the vitamin D preparations calcitriol or alphacalcidol were not included since they are not considered nutritional supplements and have a different safety profile than native vitamin D.

Study Selection Process

Figure 1. Conceptual Framework for Evaluation of the (more...)

   Figure 1. Conceptual Framework for Evaluation of the Effectiveness and Safety of Vitamin D in Relation to Bone Health

Serum 25(OH)D levels reflect cutaneous synthesis and dietary intake of vitamin D including fortified foods and supplements. For the purposes of this review, only outcomes related to bone health are considered although it is recognized that vitamin D has pleiotropic effects in the body. Outcomes assessed include fractures (related to osteoporosis or impaired mineralization), falls, and surrogate outcomes such as bone mineral density (e.g., areal or volumetric BMD), bone mineral content (BMC) and biochemical parameters such as parathyroid hormone (PTH). For women of reproductive age, calcaneal ultrasound and calcium absorption were also identified as outcomes. Note that serum 25(OH)D measurements vary depending on the particular assay used as well as the laboratory and/or operator, suggesting the need for standardization or method/laboratory-specific decision limits for vitamin D deficiency or insufficiency.

Figure 2. Modified QUOROM Flow Chart

   Figure 2. Modified QUOROM Flow Chart

Figure 2

Full text relevance screening was performed independently by two reviewers and discrepancies resolved by consensus or third party (Appendix B). Records were not masked given the equivocal evidence regarding the benefits of this practice.⁶⁵ Reasons for exclusion were noted. Relevant studies were then evaluated to determine study design and categorized accordingly for inclusion by question. The level of evidence reviewed was limited to RCTs where feasible since systematic bias is minimized in RCTs compared with all other study designs (e.g., cross-sectional, retrospective cohort). However, because of the paucity of RCT evidence addressing the association between circulating 25(OH)D concentrations and bone health outcomes, particularly in infants and young children, inclusion criteria were broadened to include single prospective cohorts, case-control, and before-after study designs for question one. Question four was restricted to existing systematic reviews to limit scope.

Quality Assessment

As part of RCT quality assessment, the Jadad scale was used (Appendix B) and scored by an experienced reviewer (Appendixes D and E). This validated scale assesses the methods used to generate random assignments and double blinding, and also scores whether there is a description of dropouts and withdrawals by intervention group.⁶⁶ The scoring ranges from 1 to 5, with higher scores indicating higher quality. An a priori threshold scheme was used for sensitivity analysis: a Jadad total score of ≥ 3 was used to indicate studies of higher quality. In addition, allocation concealment was assessed as adequate (=1), inadequate (=2) or unclear (=3) (Appendix B).⁶⁷

To assess the quality of the observational studies (prospective cohorts and case-controls), we used a grading system adapted from Harris et al.⁶⁸ Quality assessment of observational studies included variables such as representativeness of the study population, whether bias and confounding were controlled for in the study design and reported, and description of losses to followup.

An aggregate level of evidence (good, fair, inconsistent) was rated based on quantity, quality and consistency of results. As an example, for assessment of an association of circulating 25(OH)D concentrations with a bone health outcome, good evidence was defined as evidence for or against an association that was consistent across studies with at least one study graded as a higher quality study. Fair was defined by evidence sufficient to determine an association, but limited by consistency, quantity, or quality of studies (i.e., no studies graded as good). Inconsistent evidence was defined by an inability to make a conclusion for or against an association in that studies had conflicting results.⁶⁹

Qualitative Data Synthesis

Outcomes were summarized using a qualitative data synthesis for each study. A description of each study that included information pertaining to sample size and demographics, setting, funding source, 25(OH)D concentrations and assay used, intervention (form of vitamin D) and comparator characteristics, study quality, details of matching or methods of adjustment, and confounders (where applicable) were recorded and summarized in the text, and/or summary tables throughout the report. These methods were used to help generate hypotheses and to identify any heterogeneity of study populations or in the reporting of data within the published reports.

For the purpose of this review, we defined vitamin D deficiency as a serum 25(OH)D measurement below 30 nmol/L, recognizing that variable definitions have been used in the literature including values of 50 nmol/L to > 80 nmol/L (32 ng/dL), and that there is potentially large error or variability in measurement depending on the particular assay used. Similarly, vitamin D insufficiency may be defined using different values. A cutpoint of 30 nmol/L for vitamin D deficiency was used in this report to assist in classifying trials to report the results, and also when conducting subgroup analyses of trials that included vitamin D-deficient populations. In reporting individual study results, the investigator-defined definitions of vitamin D deficiency or insufficiency were noted and reported. We did not attempt to calibrate different 25(OH)D assays. As outlined in the introduction, variability may exist even when laboratories are using the same technique.

Quantitative Synthesis

For outcomes where meta-analysis was deemed appropriate, we extracted quantitative data (e.g., number of subjects in each group, mean, standard deviation) from trials, using a standardized data extraction form that included intervention characteristics (coded for vitamin D source, type of vitamin D and unit of dosing) vitamin D intake and baseline and outcome variables for all followup intervals including unit of measurement and assay used for serum 25(OH)D measurement.

Where data were only available in graph form, we attempted to extract data for the report. If relevant data (e.g., standard deviation) were not reported adequately, we contacted authors to obtain the missing data. A list of additional data received by authors is in Appendix F.

We calculated standard deviation from standard errors or 95 percent confidence intervals, and the absolute and percent change for continuous outcomes (e.g., serum 25(OH)D) from baseline and end of study data using standard formulae.

To avoid differences in the reporting of units for serum 25(OH)D concentrations (i.e., nmol/L, ng/mL, μg/dL, μg/L and ng/dL) all values were converted to nmol/L, the unit that was used for data synthesis. The conversion formula is 1 ng/mL = 2.5 nmol/L. To limit the variable reporting in vitamin D dosing (e.g., nmol, IU, ug and mg), IU was chosen as the standard unit used for meta-analysis and all other units were converted using a standard formula. The conversion formula for micrograms is 1 ug = 40 IU.

Serum 25(OH)D outcomes included absolute change values (nmol/L). Fracture outcomes were classified as vertebral, non-vertebral, hip or total fractures. BMD outcomes included absolute values (e.g., areal BMD, g/cm²), mean percent change from baseline or the difference in the mean percent change from baseline for the treatment versus comparator groups.

Followup intervals were recorded for each trial. It is common for variation to exist between trials with regard to length of followup intervals. For the purpose of meta-analyses, the most distal followup and the change between the last followup and the baseline were applied.

Statistical Analyses

For the effect measures for continuous outcomes (e.g., serum 25(OH)D concentrations) the difference in means between different treatment groups was used for the meta-analyses. The ‘difference in means’ is a standard statistic that measures the absolute difference between the mean values in the two groups in a clinical trial. Absolute change in 25(OH)D concentrations was used for quantitative pooling of 25(OH)D. For the pooling of BMD results, the percent change in BMD from baseline in the treatment versus control or placebo was used as the unit of analysis since this is clinically relevant.

For continuous outcomes, the difference in means and standard deviations were calculated for each individual study. To avoid multiple comparison issues in studies with more than one treatment arm, a weighted average (e.g., 25(OH)D) of similar groups was calculated within the study. A weighted average method was used to calculate the 25(OH)D values for the combined treatment group and combined placebo group. The difference in means was then calculated using the weighted averages for the two combined groups. This estimate, with its standard deviation was then used for the meta-analyses. The number in each group was based on intention-to-treat data; however, when these data were not available, we used what was provided in the published report.

For dichotomous outcomes (e.g., fractures, falls), studies were grouped by method of administration and type of vitamin D as we anticipated different treatment effects with (1) oral versus injectable vitamin D, (2) type of vitamin D (D₂ versus D₃) and (3) if calcium was given as a co-intervention. We used these groupings to generate pooled estimates to minimize clinical heterogeneity. The intent-to-treat group or number enrolled at the time of study was used for analyses and when unavailable, we used the number provided in the report. Combined odds ratios were generated using the number of individuals who had an event (e.g., fall or fracture) and not the absolute number of events. This was determined to be a more conservative approach to quantify the effects. For the meta-analysis of fracture and fall outcomes, we pooled studies with different treatment durations and doses.

In all cases, meta-analyses were conducted using a weighted mean method. The fixed effect model was used initially to obtain combined estimates of weighted mean differences and their standard errors. When heterogeneity (p<0.10) was present between studies, the Dersimonian and Laird random-effects method was used to obtain combined estimates across the studies.⁷⁰ The degree of statistical heterogeneity was evaluated for all analyses using the I² statistic.⁷¹–⁷³ An I² of less than 25 percent is consistent with low heterogeneity, 25 to 50 percent moderate heterogeneity, and over 50 percent high heterogeneity.⁷³ When significant heterogeneity was identified, then heterogeneity was explored through subgroup, sensitivity analyses and meta-regression analyses if appropriate. Sources of heterogeneity include methodologic as well as clinical heterogeneity. The interpretation of heterogeneity estimates requires caution especially when small numbers of trials were included.

Publication bias was explored through funnel plots by plotting the relative measures of effect (odds ratio) versus a measure of precision of the estimate such as a standard error or precision (1/standard error).⁷² Funnel plots are scatter plots in which the treatment effects estimated from individual studies, are plotted on the horizontal axis against a measure of study precision on the vertical axis. Asymmetry suggests the possibility of publication bias, although other potential causes of asymmetry exist. The degree of funnel plot asymmetry was measured by the intercept from regression of standard normal deviates against precision, with evidence of asymmetry based on p < 0.1.⁷⁴–⁷⁶

Throughout the report, vitamin D or 25(OH)D without a subscript represents either D₂ or D₃ or both isoforms. Wherever possible i.e., when reported in the particular study, the isoform is specified. All interventions are oral, unless it is specifically stated that injected vitamin D was used.

Question 1A (Part 1). Are There Specific Concentrations of Serum 25(OH)D That Are Associated With Established Vitamin D Deficiency Rickets in Infants and Young Children?

Overview of Relevant Studies

Table 1

Serum 25(OH)D Levels in Established Rickets in Infants (more...)

Table 1

Serum 25(OH)D Levels in Established Rickets in Infants and Young Children

Author (year) Country Funding	Population, N Gender Mean age (SD) Ethnicity	Matching Variables	Intervention Duration	25(OH)D isoform Measured Assay	Bone Health Outcomes	Results at baseline or diagnosis Serum 25(OH)D (nmol/L) Serum PTH (pmol/L) Serum Ca (mmol/L)
RCTs
Cesur (2003)776}	56 Infants with nutritional rickets		IG1: vit D 150,000 IU	25(OH)D3	Rickets	25(OH)D3 mean (SD) :
Turkey	36% female		IG2: vit D 300,000 IU	RIA	PTH	Stage* 1: 15.8 (6.4)
NR	10.7 (6.1) mo (range 3– 36)		IG3: vit D 600,000 IU (single dose)			Stage II: 15.4 (4.8)
	NR		2 mo			Stage III: 14.7 (3.9)
						PTH mean (SD):
						Stage I: 30 (84)
						Stage II: 34.1 (20)
						Stage III: 44.3 (25.8)
						Ca mean (SD)
						all patients
						1.9 (0.33)
Before-After Studies
Bhimma (1993)80	23 Children with rickets:		5,000–10,000 IU/d vit D3 (plus 500–1,000 mg Ca)	25(OH)D	Rickets	25(OH)D mean (SD):
South Africa	9 vit D def rickets		12 mo	CPBA		vit D deficient rickets: 9.3 (8.8)
NR	[25(OH)D < 25 nmol/L]					Ca deficient rickets: 45.5 (10)
	14 Ca def rickets					PTH: ND
	10 Phosphopenic rickets					Ca mean (SD)
	4 Healing/healed rickets					Vit D def rickets: 2.09 (0.27)
	Vit D def rickets: 56% female					Ca def rickets: 2.16 (0.28)
	NR (range 1–12 y)
	vit D def rickets (N = 9):
	6.1 (4.2) y
	NR
Elzouki (1989)81	22 Children < 2 y admitted for treatment of rickets		1–3 h/d of sunshine followed by single IM injection of 600,000 IU vit D2	25(OH)D	Rickets	25(OH)D:
Libya	37.5% female		followup median 17 d	CPBA		At diagnosis, 50% of patients had 25(OH)D > 20 nmol/L.
Public/Private	15 mo (range 3–24 mo) reported only for 16 Libyan children					Range 4–65 (graph)
	African black					PTH: ND
						Ca: ND
Garabedian (1983)78	20 Infants and children with rickets 60 Controls		IG1: 2,000 IU/d vit D2	25(OH)D	Rickets	25(OH)D mean (SD):
France/Belgium	65% female		IG2: 400 IU/kg vit D3 (single dose)	CPBA	PTH (RIA)	all patients: 11.5 (8)
NR	Mean age NR		6 mo			PTH: 2–4 X ULN (N=8); values NR
	Infants and young children (N = 15): range 4–26 mo;					Ca mean (SD)
	Older children (N = 5): range 4–12 y					All patients: 1.8 (0.27)
	80% Immigrants from North Africa, Black Africa, Turkey, Portugal, Pakistan
Markestad (1984)79	17 Children with rickets		1,700–4,000 IU vitamin D2/ d (reduced to 500–1000 IU in 3 children at 2– 4 wks)	25(OH)D	Rickets	25(OH)D median (range):
Norway	NR		10 wks	CPBA		N =9 diagnosed in summer: 21 (4.1–30.6)
Public	NR					N = 8 diagnosed in winter: 12.1 (3.8–19.4)
	11 (64.7%) Immigrants from Pakistan, Cape Verde Islands, Turkey, Morocco, Sri Lanka, and West Africa; 6 (35.3%) Norwegians					At baseline, evidence of stimulated PTH in 11/12 (serum PTH or urinary cAMP, values NR)
						Ca: ND
Case-control studies
Arnaud (1976)82	9 Children with mild (n=3), moderate (n=5) and severe (n=1) rickets	Age	Vit D 5,000 IU/d	25(OH)D	Rickets	25(OH)D mean (SD) (range):
Canada/Midwest U.S.	9 Controls		4 wks	CPBA	PTH	Mild rickets: 45 (7.5) (range 40–52.5)
Public	Rickets: 22% female					Moderate: 30 (5)
	Controls: NR					Severe: 20 (NR)
	Moderate rickets (N = 5)					Controls: 90 (30)
	Mean age 1.69 (1.03) y					Negative association between 25(OH)D and PTH (r=-0.70).
	Controls: 2.71 (1.7) y					Ca mean (SD):
	All rickets: age range 2 mo – 3.5 y					ND for mild, moderate, severe subgroups
	7 Canadian (5 First Nations, 1 West Indian black, 1 Portuguese) and 2 American (mid NW U.S.)					Stage II rickets: 2.4 (0.15)
						Age matched controls: 2.53 (0.1)
Balasubraman (2003)86	40 Children (N = 24) and adolescents (N = 19) with rickets/osteolmalacia	NR	Cases: 6,000 IU/d vit D or single dose of 600,000 IU	25(OH)D	Rickets	25(OH)D mean (SD):
India	53 controls (34 children and 19 adolescents)		3 mo	RIA		Children
NR	Rickets: 54.1% female					rickets: 50 (38.9)
	Controls: 47.0% female					controls: 61.3 (35.9), NS
	Children:					Adolescents:
	Rickets: median age 33 mo (range 11 – 120) ; Control: median 27 mo (range 6 mo – 84 mo)					rickets: 12.6 (7.1) all but one < LLN
	Adolescents:					controls: 46.0 (45.4), p<0.001
	Rickets: median 198 mo (range 168–240)					PTH: NR
	Controls: median 156 (range 120–228)					Ca mean (SD)
	Hindu/Muslim					Children
						Rickets: 2.2 (0.3)
						Controls: 2.4 (0.3) NS
						Adolescents
						Rickets: 2.1 (0.2)
						Controls: 2.3 (0.2), p=0.008
Dawodu (2005)88	38 Children with rickets	Community	NA	25(OH)D	iPTH (rickets group only)	25(OH)D median (IQR):
United Arab Emirates	50 Historical controls		NA	HPLC		Rickets: 8.0 (3.8, 15.3)
Public	Rickets: 50% female,					Controls: 43.8 (25, 64.3), p = 0.001
	Controls: 40% female					PTH showed a trend toward negative correlation with 25(OH)D (data NR)
	Rickets: 13.5 mo					Ca median (IQR)
	Controls 13.0 mo					Rickets: 2.22 (1.88, 2.35)
	Arab					Controls: 2.4 (2.25, 2.5), p= 0.001
Graff (2004)87	15 Children with rickets	Age, sex	Cases: 1,000 mg/d Ca (no vit D supplement)	25(OH)D	Rickets	25(OH)D mean (SD):
Nigeria	15 Controls (unrelated)		Treatment duration: 6 mo; Followup: 12 mo	CPBA (Nichols)	PTH (chemiluminescent immunometric assay)	significantly lower in children with rickets
NR	60% female					Rickets: 37.5 (13.5)
	Rickets: 46 (22) mo					Controls: 72.5 (11.5), p<0.001
	Controls: 47 (22) mo					PTH mean (SD)
	Rickets: 7 Muslim and 8 Christian					significantly higher in rickets group; rickets: 32 (33)
	Controls: 4 Muslim and 11 Christian					controls: 4.0 (3.1), p=0.003
						Ca mean (SD)
						Rickets: 2.13 (0.2)
						Controls: 2.4 (0.1), p <0.001
Molla (2000)85	103 Children with rickets 102 Controls	Age, sex Socio- ethnic characteristics	NA	25(OH)D	Rickets	25(OH)D mean (SD): significantly lower in children with rickets:
Kuwait	NR		NA	RIA		Rickets: 26.5 (15.5)
NR	Rickets: 14.5 (5.2) mo (range 9 mo - 8y) Controls: 15.2 (6.3) mo					Controls: 83.5 (74.75), p<0.0001
	96.1% from mothers with Hijab use					PTH: ND
						Ca, mean (SD)
						Rickets: 2.24 (0.28)
						Controls: 2.45 (0.15) p <0.0001
Oginni (1996)89	26 Children with active rickets, 90 healthy controls	Age, community	NA	25(OH)D	Rickets	25(OH)D mean (SD) (range):
Nigeria	Rickets: 50% female,		NA	RIA	PTH (radioimmunometric assay)	significantly lower in rickets group
Public	Controls: 61% female					Rickets: 36 (28), range 7–147
	Mean age NR					Controls: 69 (22), range 32–140, p<0.0002
	Children with rickets age range: 1–5 y					PTH mean (SD):
	Nigerian					higher in rickets group; Rickets: 5.9 (6.9), range 0–33.6
						Controls: 1.0 (1.2), range 0–4.1, p<0.001
						Ca (albumin corrected) mean (SD)
						Rickets: 2.06 (0.23)
						Controls: 2.35 (0.14), p<0.001
Thacher (2000)84	123 Active rickets	Age, sex if < 5 y,weight	NA	25(OH)D	Rickets	25(OH)D median (25th and 75th percentile):
Nigeria	123 Controls		NA	RIA	PTH (RIA)	Rickets: 32 (22, 40);
Public	49.6% female					< 30 nmol/L: 37%
	Mean age NR					Controls: 50 (42, 62), p<0.0001
	Rickets: median (25th and 75th percentile) age: 46 (34,63) mo					PTH median (25th and 75th percentile):
	Controls: 42 (25–70) mo					Rickets: 20 (13, 31)
	Christian/Islam:					Controls: 12 (11,16), p =0.0066
	Rickets: 82/41					Ca mean (SD)
	Controls: 57/66					Rickets: 1.93 (0.22)
						Controls: 2.24 (0.15), p<0.0001
Thacher (1997)83	37 Children with active rickets (median duration of 14 mo) 37 Healthy controls with normal weight	Age, sex	NA	25(OH)D	Rickets	25(OH)D
Nigeria	47% female		NA	RIA		Rickets: levels > LLN in 16/28 (57%); 2/28 (7%) had values < 12.5 nmol/L
NR	Rickets: 3.16 (1.53) y					Controls: ND
	Controls 3.14 (1.51) y					PTH: ND
	All Nigerian					Ca mean (SD)
						Rickets: 2.09 (0.30)
						Controls: 2.08 (0.31), NS
						55% of rickets and 51% of controls were hypocalcemic (< 2.1)

Vitamin D refers to both or one unspecified isoform; if the isoform was disclosed, it is specified as vitamin D₂ or D₃;

*

stage I rickets: early phase (serum calcium is low but serum phosphorus is normal); stage II: serum calcium normal due to compensatory hyperparathyroidism;stage III: both serum calcium and phosphorus are low ;

Ca, calcium; CPBA, competitive protein binding assay; HPLC, high performance liquid chromatography; IQR, interquartile range; IU, international units; LLN, lower limit of normal reference range; mo, month(s); NA, not applicable: ND, not done; NR, not reported; PTH, parathyroid hormone; RIA, radioimmunoassay; ULN, upper limit of normal reference range; vit, vitamin; y, year

For the purposes of this review, infancy is defined as term birth to 12 months, and young children from one to five years of age. Studies that enrolled older children were included if the majority of children were in the above age groups. For studies on established rickets in infants and young children, 13 studies met our inclusion criteria and assessed the association between serum 25(OH)D and rickets.⁷⁷–⁸⁹ Of the 13 studies, there was one RCT,⁷⁷ four before-after studies⁷⁸–⁸¹ and eight case-control studies.⁸²–⁸⁹ For the RCT, bone health outcomes included improvement in the signs and symptoms of rickets, and serum PTH levels.⁷⁷ The twelve observational studies included rickets as the bone health outcome,⁷⁸–^{84, 84}–⁸⁹ and seven of the 12 studies included assessment of serum PTH,^{78, 79, 82, 84, 87, 88} as summarized in Table 1. In all studies, children were diagnosed with rickets using clinical and radiological criteria. No studies included BMD, BMC, or fractures as outcomes.

Study characteristics including country and type of vitamin D assay are summarized in the Table 1. All studies except for one case-control study with nine participants⁸² were conducted outside of North America. The North American study was conducted at a northern latitude (Canada, U.S. Midwest). Each study examined serum 25(OH)D concentrations at diagnosis and some included followup measurements during treatment.⁷⁸–^{81, 86, 87} Six studies used an RIA assay for serum 25(OH)D assays,^{77, 83}–^{86, 89} six studies used a CPBA method,⁷⁸–^{82, 87} and one study used an HPLC technique.⁸⁸ We report, in this section, baseline measurements at diagnosis or pre-treatment.

Population characteristics. Children with rickets ranged in age from as young as two months up to 14 years, with most children between 24 and 36 months. In the studies that reported ethnicity, virtually all children were non-white except for two subjects in the one North American study.⁸² The sample sizes ranged from nine⁸² to 123 participants,⁸⁴ with an average of 41. In 12 of the 13 studies, gender was mixed.

Outcome characteristics. For all studies, the diagnosis of rickets was ascertained by radiographic and clinical evidence.⁷⁷–^{87, 89} Serum PTH was measured in seven studies using either RIA or chemiluminescent immunoassays.^{78, 79, 82, 84, 87}–⁸⁹ No study evaluated BMC, BMD or fractures.

Study quality. The study quality of the RCT,⁷⁷ four before-after and eight case-control studies ranged from poor to fair with the RCT scoring 1/5 on the Jadad scale (in relation to randomization for treatment).

Qualitative synthesis of individual study results. Six studies reported a mean^{77, 78, 80, 85} or median^{79, 88} serum 25(OH)D concentration < 27.5 nmol/L associated with rickets. These studies included measurements by RIA,^{77, 85} CPBA⁷⁸–⁸⁰ or HPLC.⁸⁸ Five studies reported that children with rickets had a mean 25(OH)D concentration above 27.5 nmol/L (range of means 36 – 50 nmol/L),^{82, 84, 86, 87, 89} and the other two studies reported at least some children with serum levels above this value.^{81, 83} While 25(OH)D assays differed across the studies, these results suggest that the serum 25(OH)D concentration associated with rickets may be much higher than previously thought. In one study, deficient dietary calcium was the etiology for rickets⁸³ whereas in another study, a mean dietary calcium intake of < 300 mg/d did not alter the Odds Ratio (OR) for rickets.⁸⁴ Given the uncertainty of the dietary calcium measurement, it remains unclear whether the specific concentration of serum 25(OH)D consistent with rickets is confounded by dietary calcium.

In the studies that reported serum PTH, values in children with rickets were elevated above the normal range.^{78, 79, 82, 84, 87, 89} One study confirmed a negative relation of PTH with 25(OH)D concentrations (r = -0.70),⁸² when cases and controls were analyzed together.

The majority of studies included in this review were from developing countries where dietary calcium intake is low. Low dietary calcium can confound 25(OH)D status and is a major limitation of the studies since some cases of rickets may be attributable to a calcium deficiency. Another limitation is the paucity of studies in children with rickets in North America. The specific concentrations of serum 25(OH)D associated with rickets in North America is uncertain, given the lack of studies in populations with dietary calcium intake similar to North American diets, as well as the different methods used to determine 25(OH)D concentrations. A better understanding of the inter-relationship between 25(OH)D concentrations, calcium and rickets would improve the specific values of 25(OH)D to be used as a biomarker in the diagnosis and treatment of rickets. Only studies of established rickets were included, and other RCTs have evaluated specific 25(OH)D concentrations in relation to the development of rickets. In a rickets prevention study in China, Specker et al. found that 25(OH)D concentrations above 30 nmol/L appeared to prevent rickets in infants with or without vitamin D deficiency at birth.⁹⁰

Summary. Circulating 25(OH)D levels associated with established rickets in infants and young children

Quantity: Six studies (one RCT, three before-after and two case-control studies) reported mean or median 25(OH)D concentrations < 30 nmol/L in children with rickets whereas the other studies reported mean or median values above 30 nmol/L and up to 50 nmol/L. In seven of eight case-control studies, serum 25(OH)D values were lower in the children with rickets compared with controls.

Quality: The study quality of the RCT, four before-after and eight case-control studies ranged from poor to fair (with the RCT scoring 1/5 on the Jadad scale).

Consistency: There is fair evidence for an association between low serum 25(OH)D and established rickets, regardless of assay type (RIA, CPBA, HPLC). There is inconsistent evidence to determine if there is a threshold concentration of serum 25(OH)D above which rickets does not occur.

Question 1A (Part 2). Are Specific Circulating Concentrations of 25 Hydroxyvitamin D [25(OH)D] Associated With Bone Health Outcomes in Infants?

Overview of Relevant Study Characteristics and Results

Infancy is defined by the Institute of Medicine as including two subcategories: birth to 6 months and 6 to 12 months.⁴ Seven studies included infants 12 months or younger and assessed the association between serum 25(OH)D and bone health outcomes.⁹¹–⁹⁷ Of the studies, there were three RCTs, two in breast-fed infants^{92, 93} and one in formula-fed infants,⁹¹ and four case-control studies.⁹⁴–⁹⁷

Table 2

Serum 25(OH)D and Bone Health Outcomes in Infants

Table 2

Serum 25(OH)D and Bone Health Outcomes in Infants

Author (year) Country Funding	Population, N Gender Mean Age (SD) Ethnicity	Intervention Duration	Serum 25(OH)D Assay Time points	Bone Health Outcomes	Results	Jadad Score
RCTs
Greer (1982)93	18 Healthy term infants exclusively breast-fed	IG1: vit D2	25(OH)D	PTH (RIA)	Serum 25(OH)D mean nmol/L	3
U.S.	IG1 9; CG 9	400 IU/d	CPBA	distal L radius BMC (SPA)	Baseline: no significant difference between groups
Public	At 9 mo, 6/13 and at 12 mo, 3/13 enrolled infants were still breastfeeding	CG: placebo	Measured at baseline, 12 and 26 wks	Measured at 3, 6, 12, 26, 40 and 52 wks	12 wks:
	66% female	12 wks (double blind); (unblinded to investigator at 3 mo); supplements continued until weaned			IG1:95* (graph)
	0 d (recruited at birth)	At 6 mo, unblinded to mother, and placebo group began to received daily vit D2 400 IU/d			CG: 50
	17 Caucasian 1 Asian-Indian	followed to 1 y			26 wks:
					IG1: 81.8
					CG: 32.3
					PTH: no significant difference between groups (data NR)
					BMC mean (SEM) mg/cm
					12 wks: IG1 79 (3); CG 64 (3), p < 0.003
					26 wks: IG1 70 (6); CG 75 (5), NS
					52 wks: IG1 108 (20); CG 120 (19) (CG receiving vit D for 6 mo)
Greer (1989)92	46 Healthy term born infants born to mothers willing to breast-feed for 6 mo, 12 additional controls (formula fed infants)	IG1: 400 IU/d D2	25(OH)D and each isoform measured	PTH (RIA)	Total serum 25(OH)D mean (SD)	4
USA	46% female	CG: placebo	HPLC	distal L radius BMC (SPA)	At birth:
Public	NR (range 37 to 40 wk gestation)	6 mo, starting at birth	Measured at birth, 1.5, 3 and 6 mo	Measured at 1.5, 3 and 6 mo	IG1: 59.7 (11.8)
	All infants: Caucasian mothers; fathers: 1 black, 1 American Indian, others Caucasian				CG: 58.8 (19.1)
					6 mo:
					IG1: 92.4 (29.7)
					CG: 58.8 (24.9), p < 0.01
					PTH: no significant difference between groups
					BMC mean (SD) mg/cm:
					No significant difference between groups at 1.5 and 3 mo. At 6 mo, CG was significantly greater than IG1: IG1 89.5 (12.5) vs. CG 101.0 (17.9), p<0.05 However, change in mean BMC from 1.5 to 6 mo was not different between groups.
Zeghoud (1997)91	80 Healthy neonates, and their mothers; after initial measurements, infants were divided into 3 groups based on serum 25(OH)D (≤ or > 30 nmol/L) and PTH ≤ or > 60 ng/L)	IG1: 500 IU IU/d D2	25(OH)D	iPTH (RIA)	Serum 25(OH)D mean (SD) Baseline total sample: 29.5 (13.8); (range 10–80)	1
France	NR	IG2: 1000 IU/d D2	CPBA	Measured at 3–6 d, 1 mo, 3 mo	51/80 (63.7%) ≤ 30 nmol/L
NR	NR (range: 3 to 6 d)	Starting at 3–6 d after birth	Measured at 3–6 d, 1 mo, 3 mo.		Serum iPTH was negatively correlated with 25(OH)D (r = 0.45, p < 0.001)
	From birth to 3 mo, 28 (35%) excluded, some (< 10) due to digestive problems	All infants fed formula with mean (SD) 426 (46) IU vitamin D3/L			In neonates with 25(OH)D < 16 nmol/L, iPTH was significantly higher: mean (SD) 70 (30) pmol/L than those born with 25(OH)D > 30 nmol/L
	European				Infants with high iPTH (> 60 ng/L) were born to mothers with 25(OH)D <30 nmol/L.
					Mean baseline 25(OH)D by group**:
					Group 1 (N = 14): 25(OH)D ≤ 30 nmol/L and iPTH > 60 ng/L: 17.9 (7.8)
					Group 2 (N = 36): 25(OH)D ≤ 30 nmol/L and iPTH < 60 ng/L: 22.7 (6.5)
					Group 3 (N = 29) 25(OH)D > 30 nmol/L and iPTH < 60 ng/mL: 43.7 (10.6)
					At 1 mo, all 3 groups (pooled vit D doses):
					mean serum 25(OH)D was significantly increased and there was no significant difference between groups.
					Group 1: 53.1 (12)
					Group 2: 59.8 (17.7)
					Group 3: 59.2 (11.4)
					At 1 mo, iPTH decreased and there was no significant difference between groups (pooled doses).
					At 3 mo, mean 25(OH)D for total sample (pooled doses) was 69 nmol/L; highest value 92.5 nmol/L.
					IG1 (500 IU D2)
					For group 1, at 1mo (45.5 nmol/L) and 3 mo (56.1 nmol/L), serum 25(OH)D values were significantly lower than the other 2 groups receiving same dose, and lower than all groups receiving 1,000 IU/d.
					Serum iPTH remained elevated in 14.3% of infants in group 1 after 1 mo, and mean PTH was significantly higher than those of other grps at 1 and 3 mo.
					IG2 (1,000 IU D2)
					Serum iPTH was similar among the 3 groups receiving 1000 IU/d at 1 mo. PTH declined in all grps and did not change between 1 and 3 mo.
					Change in serum 25(OH)D (3 mo) was not significantly different between the 3 groups.
Case-control studies
Okonofua (1986)94	21 Healthy term born infants		25(OH)D	PTH (RIA-midportion)	Serum 25(OH)D mean (SD) (nmol/L):
UK	NR		Cord and maternal sampling	fractures during birth	Lower in Asian vs. white term born infants (p<0.01)
NR	NR		CPBA		White: 15 (5) (range 9–39)
	10 Caucasian (47.6%),		Measured at baseline		Asian: 6 (4) (range < 5 – 20)
	11 Asian (52.4%)				Mean (SD) serum PTH (pmo/L):
					Higher in Asian vs. white infants (p < 0.05)
					White: 55 (6)
					Asian: 44 (7)
					Maternal 25(OH)D in white mothers was 30 (11) nmol/L and in Asian mothers was 15 (10) nmol/L serum PTH was higher in Asian mothers.
					25(OH)D levels in mothers were significantly higher than neonatal levels; the two were correlated (r=0.60).
					fractures during birth: 0
Bougle (1998)97	82 Healthy term born infants (also 44 preterm)		25(OH)D	LS BMD and BMC (DXA)	Full term infants:
France	NR		Assay NR		Serum 25(OH)D mean (SD) nmol/L (range) 75 (52.5) (10–292.5)
NR	Term 40 wks (range 37–42)		At or following hospital discharge		Full term infants:
	Asian				25(OH)D negatively related to BMD (r =-1.7, p=0.02) and to BMC in full term (r =-0.04, p=0.02), in a simple regression analysis but not related to BMC or BMD in a multiple regression analysis.
Namgung (1998)95	71 Healthy term infants,		25(OH)D	iPTH (Allegro RIA)	Serum 25(OH)D mean (SD) (nmol/L):
Korea	37 born in summer,		Measured in cord samples	Whole body BMC (DXA) measured before 3 d of age	Winter born infants had lower 25(OH)D than summer born (p<0.001).
Public	34 born in winter		HPLC		Winter born: 26.8 (19.0)
	Winter 38% female		Winter 26.8 (19.0)		Summer born: 75.0 (24.0)
	Summer 59% female		Summer 75.0 (24.0)		% of infants with levels < 27.5 nmo/L
	Mean (SD) gestational age:				Winter born: 97%
	Winter: 38.3 (0.7) wks				Summer born: 47%
	Summer: 38.3 (0.8) wks,				No differences were observed for PTH.
	range 37 – 41 wka				Serum PTH geometric mean (range):
	Korean				Winter born: 5.8 (2.8 – 11.9)
					Summer born: 5.1 (1.8 – 14.6), NS
					Winter born had 8% lower whole body BMC than summer born (p = 0.0002).
					BMC LSM (SD) (g/cm):
					Winter born: 86.7 (7.7)
					Summer born: 93.9 (7.8)
					Whole body BMC correlated positively with serum 25 (OH)D (r=0.243, p=0.047).
					Maternal 25(OH)D was lower in winter than summer: 24 (13) vs. 43 (18), p < 0.001.
Park (1998)96	35 Healthy term born infants born in winter,		25(OH)D	iPTH (Allegro RIA)	Serum 25(OH)D mean (SD) nmol/L:
Korea	18 exclusively breast-fed, 17 formula-fed with 400 IU vitamin D		HPLC	LS BMC and BMD (DXA)	Mean was lower in breast-fed vs. formula-fed infants, p = 0.001
NR	enrolled at ages 2 – 5 mo		Measured at recruitment (ages 2 – 5 mo)		Breast-fed: 39.9 (28.2)
	Breast-fed: 28% female;				Formula-fed: 72.5 (22.2)
	Formula-fed: 47% female				% with 25(OH)D < 28 nmol/L
	Breast-fed: 3.3 (1.2) mo;				Breast-fed: 8/18 (44%)
	Formula-fed: 3.6 (1.1) mo				Formula-fed: 1/17 (6%), p=0.01
	Korean				Serum PTH mean (SD) (ng/L)
					Breast-fed: 14.8 (6.93)
					Formula-fed: 11 (5.47), NS
					LS BMD no difference between breast-fed (N = 14.18) and formula-fed infants (N = 14/17) (data NR)
					LS BMC mean (g/cm) (SD)
					No difference between groups
					Breast-fed: 0.62 (0.2)
					Formula-fed: 0.65 (0.2)
					25(OH)D did not correlate with BMC (r=0.173, p=0.39, N=28).

refers to both or either isoform of 25(OH)D (isoform not specified); if reported, the isoform is specified.

+

Jadad score out of 5; for all RCTs in the table, allocation concealment was assessed as “unclear”.

*

SEM provided in graph but not estimable

**

1/80 infants did not clearly fit into any category and had findings suggestive of transient congenital hypoparathyroidism

AC, allocation concealment: BMC, bone mineral content; BMD, bone mineral density; DXA, dual X-ray absorptiometry; iPTH, intact PTH; IU, international units; LS, lumbar spine; LSM, least squares mean; mo, months; NR, not reported; NS, not significant; PTH, parathyroid hormone; RIA, radioimmunoassay; SD, standard deviation; SPA, single photon absorptiometry; y, year(s)

For the three RCTs, bone health outcomes included BMC^{92, 93} and serum PTH levels⁹¹–⁹³ (Table 2). No RCTs reported results of BMD or evaluated fracture incidence. Four observational studies reported BMC,⁹⁵–⁹⁷ BMD,^{96, 97} fractures⁹⁴ or PTH (Table 2).⁹⁴–⁹⁶

Study characteristics. Of the three RCTs, two were conducted in the U.S.^{92, 93} Both of these trials randomized human milk-fed infants to receive vitamin D₂ supplementation (400 IU/d) or placebo. One U.S. RCT was six months in duration,⁹² and the other was 26 weeks long at which time the placebo group were started on supplementation, and both groups were followed until 52 weeks.⁹³ The RCT by Zeghoud et al. was three months in duration, and randomized infants to receive either 500 or 1000 IU/d D₂.⁹¹ The 25(OH)D assays varied, with two studies using a CPBA method^{91, 93} and one using HPLC.⁹²

None of the four case-control studies were conducted in North America (Table 2). Outcomes were assessed at birth in three studies^{94, 95, 97} and at two to five months of age in the other.⁹⁶ One study measured circulating 25(OH)D by CPBA,⁹⁴ two studies used HPLC,^{95, 96} and the fourth study⁹⁷did not report the method.

Population characteristics. For the three RCTs, the age at enrolment was within a few days of birth.⁹¹–⁹³ The sample sizes ranged from 18 to 80 infants, without a predominance of male or female gender. In all three studies,⁹¹–⁹³ participants had to be healthy and free of conditions known to affect calcium metabolism. Mean vitamin D and calcium intake were not reported in any of the studies, although maternal behavior related to breast feeding was reported in all studies. Baseline 25(OH)D concentrations are summarized in Table 2.

For the case-control studies, three studies evaluated infants at birth or within the first few days of birth,^{94, 95, 97} and one study evaluated infants at two to five months of age.⁹⁶ The sample sizes ranged from 21 to 82 infants with sub-categorization as to ethnicity,⁹⁴ term born,⁹⁷ season of birth,⁹⁵ or feeding type.⁹⁶ In all case-control studies, participants had to be healthy and free of conditions known to affect calcium and bone metabolism. Data on dietary vitamin D or calcium intake plus exposure to sunshine were only relevant for the study that evaluated two to five month old infants,⁹⁶ and these data were not reported.

Covariate/confounders. No relevant covariates or effect modifiers were controlled for in the RCTs. In one RCT, baseline 25(OH)D concentrations were used to divide the study cohort into three subcategories⁹¹ (Table 2). Seasonal effects were examined in one study.⁹² For case-control studies, matching on gestational age at birth and gender was not reported. Only one study adjusted for weight when evaluating the relation between 25(OH)D and whole body BMC.⁹⁵

Outcome characteristics. For the RCTs, BMC of the distal radius was measured by single photon absorptiometry,^{92, 93} and PTH was measured using RIA.⁹¹–⁹³

For the case-control studies, BMC (whole body or spine) and BMD were measured using dual-energy x-ray absorptiometry (DXA).⁹⁵–⁹⁷ PTH was measured using RIA techniques.⁹⁴–⁹⁶ Although all studies used RIA techniques to measure PTH, these may have varied in antibody specificity and measurement of PTH fragments.⁹⁸

One case-control study reported fracture incidence⁹⁴ although the methodology was not reported.

Study quality. For the RCTs, one trial each scored 1/5,⁹¹ 3/5⁹³ and 4/5⁹² on the Jadad scale. The four case-control studies were of fair quality.

Qualitative synthesis of individual study results. Of the two RCTs measuring BMC of the distal radius, one study showed transient elevation in BMC at 12 weeks of age in the supplemented group (with serum 25(OH)D concentrations of 95 nmol/L) compared to the placebo group (with 25(OH)D concentrations of 50 nmol/L).⁹³ However, by 26 weeks there was no significant difference in BMC between the placebo and vitamin D₂ supplemented infants who continued to have higher serum 25(OH)D levels. In a second trial by Greer,⁹² no difference in BMC was observed at 3 months in vitamin D₂ supplemented or unsupplemented human milk-fed infants despite 25(OH)D concentrations of 97 nmol/L in the intervention group compared to 39 nmol/L in the control group. At six months, the control group had higher absolute BMC and was also noted to have higher levels of the (unsupplemented) D₃ isoform. However, the change in BMC from 1.5 to 6 months was not significantly different in the two groups.

Two case-control studies measured BMC and BMD of the lumbar spine (L1–4).^{96, 97} One study observed a negative correlation between 25(OH)D (levels ranging from 10 to 292 nmol/L) and spine BMC and BMD at birth but no relation was observed in regression analyses that included postnatal age and serum calcium.⁹⁷ The other study⁹⁶ did not find a difference in spine BMC at two to five months of age when a group of human milk-fed infants with an average 25(OH)D serum level of 40 nmol/L were compared with a group of formula-fed infants with an average 25(OH)D of 73 nmol/L. 8/18 infants in the human milk-fed group and 1/17 in the formula-fed group had a serum 25(OH)D level < 28 nmol/L; there was no correlation of BMC with serum 25(OH)D concentration. The one study that measured whole body BMC reported a positive relation between 25(OH)D and BMC.⁹⁵ The values for 25(OH)D in this study were on average 27 nmol/L for winter born and 75 nmol/L for summer born who had eight percent higher whole body BMC at birth.

Overall, for BMC measurements reflecting mainly cortical bone, including whole body and radial assessments, two of three studies showed a positive association between 25(OH)D concentrations with BMC, one measuring whole body BMC and one showing a transient increase in distal radial BMC at 12 but not 26 weeks.^{93, 95} Of the two studies examining predominantly trabecular bone (lumbar spine),^{96, 97} one showed a negative correlation between 25(OH)D and BMC and BMD at birth that was not evident after using multiple regression; ⁹⁷ the other did not demonstrate any association.

Of the two RCTs reporting PTH levels, one study did not observe differences in PTH between vitamin D₂ supplemented and non supplemented infants at 1.5 to six months of age.⁹² Both groups were characterized by mean serum 25(OH)D levels above 30 nmol/L (measured by HPLC). At all timepoints, 25(OH)D values were higher in the supplemented group (range of means from 75.6 to 97.2 nmol/L compared to means of 39.4 to 58.8 nmol/L in the unsupplemented group). In the other RCT, PTH declined in all groups from birth to three months of age while 25(OH)D concentrations increased to at least 46 nmol/L (measured by CPBA).⁹¹ In that study, all neonates who had abnormally high PTH had serum 25(OH)D < 30 nmol/L. In a case-control study, serum PTH was not different among winter and summer born infants with mean serum 25(OH)D of 27 and 75 nmol/L respectively (measured by HPLC).⁹⁵ Similarly, human milk-fed infants with a mean 25(OH)D concentration of 40 nmol/L did not have different serum PTH values than formula-fed infants with a mean 25(OH)D concentration of 73 nmol/L (measured by HPLC).⁹⁶ Lastly, Asian infants had significantly higher PTH concentrations and lower 25(OH)D concentrations of 5 to 20 nmol/L (mean 6, SD 4) when compared to Caucasian infants characterized by serum 25(OH)D concentrations of 9 to 39 nmol/L (mean 15, SD 5) (measured by CPBA).⁹⁴ Overall, these five studies suggest that PTH is inversely associated with serum 25(OH)D concentrations at lower 25(OH)D concentrations but there was inconsistent evidence for a threshold that may exist somewhere above 27 nmol/L (measured by CPBA). Variable evidence for a threshold may be in part due to the different assays used, both to measure serum PTH and serum 25(OH)D.

Of the studies examining a relation between 25(OH)D and bone health outcomes, most had small sample sizes and the baseline 25(OH)D was variable ranging from deficient values around the limitation of detection to values above 27 nmol/L. In studies with repeated measurements, the baseline 25(OH)D was not considered as an effect modifier in evaluating the relation between 25(OH)D and bone health outcomes. The three included RCTs used vitamin D₂ supplementations and therefore conclusions cannot be drawn regarding supplementation with the D₃ isoform. Lastly, a definitive conclusion as to whether a specific concentration of 25(OH)D is associated with an elevated PTH (secondary hyperparathyroidism) is not possible given the evidence put forth to date. Additional studies are required to define a threshold concentration of 25(OH)D below which serum PTH levels rise. This will require not only standardization of 25(OH)D assays but also PTH assays.⁹⁸

Summary. Serum 25(OH)D levels and bone health outcomes in infants

Quantity: Of the two RCTs examining BMC, one demonstrated no benefit of higher serum 25(OH)D on radial bone mass while the other showed a transient increase of BMC compared to the unsupplemented group at 12 weeks but not 26 weeks. Of the three case-control studies, whole body BMC was positively related to and lumbar spine negatively related to serum 25(OH)D concentrations. Based on two RCTs and three case-control studies, a rise in PTH was either not observed with 25(OH)D concentrations above 27–30 nmol/L or occurred at a lesser rate than at lower values, suggesting a threshold value may exist somewhere above 27 nmol/L.

Quality: The three RCTs were of fair to high quality (two of the three RCTs had a Jadad score of ≥ 3/5) and the four case-control studies were of fair quality.

Consistency: There is inconsistent evidence for an association between a specific concentration of serum 25(OH)D and the bone health outcome BMC in infants. Overall, there is fair evidence that PTH is inversely associated with serum 25(OH)D concentrations at lower 25(OH)D concentrations, but there was inconsistent evidence for a threshold that may exist somewhere above 27 nmol/L (measured by CPBA).

Question 1A (Part 3). Are Specific Circulating Concentrations of Serum 25 Hydroxyvitamin D [25(OH)D] Associated With Bone Health Outcomes in Older Children and Adolescents?

Definition of study populations. The Institute of Medicine defines early childhood as ages 4 though 8 years, and puberty/adolescence as ages 9 through 13 years, and 14 through 18 years.⁴ Grouping by age for the purpose of this report were based on the study populations. In this section, children six years of age or older who had not yet entered puberty were included, and adolescence (marked by the onset of puberty) was defined by the presence of at least Tanner Stage 2 for sexual development.⁹⁹ The age groups in the included studies for this section were: 6–10 years,¹⁰⁰ age 9 years,¹⁰¹ 8 – 10 years,¹⁰² 9 –15 years,¹⁰³ 15–16 years,¹⁰⁴ 10 – 17 years,¹⁰⁵ and 10 – 18 years.¹⁰⁶

Study characteristics. Three studies that included older children (one RCT,¹⁰² one prospective cohort¹⁰¹and one before-after study¹⁰⁰) assessed the association between serum 25(OH)D concentrations and bone health outcomes.

Four studies in adolescents assessed the association between 25(OH)D levels and bone health outcomes.¹⁰³–¹⁰⁶ There were two cohort studies,^{103, 104} one case-control study¹⁰⁶ and one RCT.¹⁰⁵ The first cohort evaluated the association between serum 25(OH)D levels and lumbar spine and femoral neck BMD/bone mineral apparent density (BMAD) at baseline and 3 years.¹⁰³ The second cohort study evaluated the seasonal variation in serum 25(OH)D concentrations and its relation to intact (i) PTH levels over an 18 month period.¹⁰⁴ El Hajj Fuleihan¹⁰⁵ evaluated the effect of low (1,400 IU/week) and high (14,000 IU/week) dose vitamin D₃ on areal BMD and BMC of the lumbar spine, hip, forearm, and total body and body composition. Marwaha¹⁰⁶ evaluated 25(OH)D concentrations in 5,137 children and adolescents (aged 10–18 years) from Northern India and the association with serum PTH, ionized calcium and BMD of the forearm and calcaneus, with stratification by upper and lower socioeconomic status.

Bone health outcomes - ascertainment. For the studies on older children, PTH was measured by an immunoradiometric assay that detects the mid-region of the molecule,¹⁰² and distal radial BMC was measured by single-photon absorptiometry (SPA).¹⁰² Javaid¹⁰¹ measured whole body and lumbar spine BMC and areal BMD by DXA, and calculated an apparent volumetric BMD at nine years of age in relation to maternal third trimester 25(OH)D status. Rajakumar¹⁰⁰ evaluated the association between serum 25(OH)D concentrations, serum PTH and markers of bone turnover.

For adolescents, lumbar spine BMD, femoral BMD, and lumbar spine bone mineral apparent density (BMAD) was measured by DXA¹⁰³ and iPTH by immunoradiometric assay.¹⁰⁴ Fuleihan measured areal BMD and BMC at the lumbar spine, hip and forearm, and total body and lean body mass by DXA.¹⁰⁵ Marwaha¹⁰⁶ evaluated forearm and calcaneal BMD using peripheral DXA and PTH with an immunoradiometric assay.

There were no studies that assessed the association between serum 25(OH)D concentrations and fractures in older children or adolescents.

For assessment of 25(OH)D levels, different methods were used depending on the study. These included radioimmunoassay or radioimmunometric methods in three studies,^{101, 103, 106} and CPBA in three studies.^{100, 104, 105}

Population characteristics. For older children, ages ranged from eight to ten years in two studies with mixed gender.^{101, 102} Included subjects were aged 6 – 10 years in the Rajakamar study who exhibited a combination of pre- and early pubertal status (33/42 pre-pubertal Tanner stage I).¹⁰⁰ Eligibility criteria for two studies required that participants be healthy, without co-morbidities.^{100, 102} The prospective cohort study by Javaid did not state whether children with co-morbidities were excluded. The mean dietary intake of calcium/vitamin D was reported in two studies.^{100, 101}

Table 3

Serum 25(OH)D Levels and Bone Health Outcomes in Older (more...)

Table 3

Serum 25(OH)D Levels and Bone Health Outcomes in Older Children and Adolescents

Author (year) Country Funding	Population, N Attrition Gender Mean age Ethnicity	Intervention Duration	25(OH)D Assay	Bone Health Outcomes	Results	Jadad AC
RCTs
Ala- Houhala (1988)102	60 Children, 8 – 10 y old	IG1:Vit D2	25(OH)D	PTH (midregion 44–68, RIA)	Serum 25(OH)D mean (SD) nmol/L	1
Finland	IG1: 30; CG: 30	400 IU 5–7×/wk	Measured at baseline (1st winter) mid-study (autumn), and end of study (2nd winter)	distal radius BMC (SPA)	Baseline (winter):	Unclear
Public	Excluded:	CG: placebo	CPBA		IG1: 49.3(19.0) vs. CG: 46 (15.5)
	IG1 6; CG 3	13 mo			Mid-study (autumn):
	% female:				IG1: 78 (24.3) vs. CG 59 (17.8)
	IG1 62%;				End-of-study (winter):
	CG 48%				IG1: 71.3 (23.4) vs. CG 43.3 (19.5), p < 0.01
	NR; range 8–10y				Baseline serum PTH mean (SD) pmol/L:
	Caucasion				IG1: 40 (20); CG 39 (19) (NS)
					No difference between groups in PTH at 13 mo
					No difference between groups in distal radius BMC at 13 mo
Fuleihan (2006)105	179 children and adolescent girls (34 pre-menarcheal and 134 post-menarcheal)	IG1: 1,400 IU D/wk	25(OH)D	BMD and BMC LS, forearm, total body DXA (Hologic 4500A)	25(OH)D mean (SD) nmol/L	4
Lebanon	IG1: 62	IG2:14,000 IU D/wk	Measured at baseline, 6 mo, 1y		baseline:	Unclear
Private	IG2: 59	CG: Placebo	CPBA (Incstar, DiaSorin)		IG1: 35 (22.5)
	CG: 58	1y			IG2: 35 (20.0)
	Lost to follow up or discontinued: 11				CG: 35(17.5)
	100% female				1y:
	10–17 y				IG1: 42.5 (15)
	Middle Eastern				IG2: 95 (77.5)
					CG: 40 (20.0)
					Covariates: percent change in bone area, percent change in lean mass
					Significant association between baseline serum
					25(OH)D and:
					LS BMD (r=0.16, p=0.033),
					Femoral neck (r=0.17, p=0.028), and
					Radius BMD levels (r=0.24, p=0.002)
					Radius BMC levels (r=0.16, p=0.033)
					Largest increases in bone mass in IG2 (high dose) subjects with lowest 25(OH)D levels at baseline
Prospective Cohort Studies
Guillemant (1999)104	175 Healthy adolescent boys from a jockey training center	NA	25(OH)D	iPTH (immunoradiometric assay, Nichols)	25(OH)D mean (SD)
France	100% male		Measured after summer (Sept– Oct) and after winter (March–April)		Post-summer 58.5 (10)
NR	Range 13 y 5 mo to 16 y 1 mo		CPBA		Post-winter 20.6 (6.0), P=0.0001
	Caucasion				iPTH negatively correlated with 25(OH)D, non-linear, (p <0.001, r=-0.504)
					At > serum 25(OH)D > 83 nmol/L, iPTH plateau occurred at 2.48 pmol/L
					seasonal variation in mean (SD) iPTH: summer 2.76 (0.97) vs. winter 4.20 (1.21) pmol/L
Javaid (2006)101	198 Children with known maternal 25(OH)D status in third trimester (original cohort: children born to 596 white women in a study of maternal nutrition and fetal growth 1991- 1992 )	NA	25(OH)D	Total body and lumbar spine BMC and areal BMD calculated volumetric BMD (DXA Lunar DPX-L)	Maternal serum 25(OH)D in late pregnancey:
U.K.	9 y old		Measured in mothers in third trimester		18% had serum 25(OH)D levels < 27.5 nmol/L and
Public	Caucasion		RIA (IDS)		31% had levels 27.5–50 nmol/L
					Mothers with lower 25(OH)D during pregnancy had children with reduced total body (r=0.21, p=0.0088) and lumbar spine BMC (r=0.17, p=0.03). Adjustment for height did not weaken the relationship between total body BMC and 25(OH)D; Volumetric LS BMD was not associated with maternal 25(OH)D.
					adjusted for age of child
Lehtone-Veromaa (2002)103	191 Healthy adolescent girls	NA	25(OH)D	LS BMD and BMAD	25(OH)D mean (SD) nmol/L
Finland	15 (7.9%) dropped out during the 3 y (final N=171)		baseline, 1 and 3 y	FN BMD and BMAD	baseline: 34.0 (13.2) (winter)
Public	100% female		RIA (DiaSorin)	DXA (QDR 4500C Hologic)	1 y: 33.2 (11.1)
	12.9 (1.7) y, range 9–15 y				3 y: 40.6 (15.8)
	Caucasian				Baseline 25(OH) D correlated with Δ LS BMD (r=0.35, p < 0.001) and Δ FN BMD (r=0.32, p < 0.001)
					Baseline 25(OH)D correlated with Δ LS BMAD (0.35, p < 0.001) and Δ FN BMAD (0.24, p < 0.002)
					Adjusted for: baseline reproductive y, bone mineral values, increases in height and weight, mean intake of calcium and mean amount of physical activity Significant correlation between baseline 25(OH)D and Δ 3-y adjusted LS or FN BMD and BMAD.
					Difference in mean 3-y Δ LS BMD between group with baseline 25(OH)D<20 nmol/L and group with baseline 25(OH)D ≥37.5 was 4%.
Case-Control Studies
Marwaha (2005)106	5137 Healthy school children	NA	25(OH)D	BMD (distal forearm and calcaneum) using DXA (Lunar PIXI-1.34)) measured in subset N = 555	Serum 25(OH)D mean (SD): 29.5 (18)
India	3089 from Lower Social Economic Status (LSES), 2048 from Upper Social Economic Status (USES)		RIA	iPTH (immunoradiometri c assay, DiaSorin) N = 740	LSES: 26 (1); USES: 34 (1) 25(OH)D < 22.5 nmol/L: 35.7%; LSES 42.3% vs. USES 27%, p < 0.01
NR	% female:		Measured in subset N = 740		Prevalence of clinical vitamin D deficiency (defined by genu varum or genu valgum): LSES 11.6% vs. USES 9.7%, p=0.07
	LSES: 65.1%				Forearm mean BMD significantly higher (p<0.01) in USES group compared to LSES
	USES: 52.7%				BMD adjusted for height and weight
	Mean age NR				Serum Ca no significant difference between groups but dietary calcium intake lower in LSES group
	Range 10 – 18 y				No significant correlation between BMD and serum 25(OH)D in either group
	Indian				Significant negative correlation between PTH and 25 (OH)D, r=0.020, p<0.01
Before-After Studies
Rajakumar (2005)100	42 Healthy 6 – 10 y olds	Vit D 400 IU/ d (isoform not specified)	25(OH)D	iPTH (Immulite iPTH chemiluminescent assay)	Serum 25(OH)D mean (SD) nmol/L
U.S.	Tanner stage I/II (81% I)	1 mo	Measured at baseline and 1 mo		baseline: 60.0 (26.3)
Public	Skin type III/IV (81% IV)		CPBA (Nichols Advantage chemiluminescence)		49% < 50
	Vit D dietary intake: mean (SD) 277 (146) IU/d				71% < 75
	16/41 (39%) dietary intake < 200 IU/d				Group 1 = 25(OH)D < 50 nmol/L at baseline: 38.5 (8.0)
	2 withdrew for personal reasons				Group 2 = 25(OH)D > 50 nmol/L at baseline: 80.3 (20.5)
	34% female				1 mo (total group): 68.8 (18.8)
	8.9 (1.2) y (range 6 –10 y)				Group 1: 57.5 (16)
	African American				Group 2: 79.5 (14.5)
					Increase in serum 25(OH)D was observed only in group 1
					7/39 (18%) of group 1 continued to have a level < 50 nmol/L after 1 mo of supplementation
					Negative correlation between 25(OH)D and PTH at baseline (r = -0.325, p = 0.038)
					Inflection point for PTH started at 25(OH)D ~ 75 nmol/L
					iPTH mean (SD) pmol/L
					Baseline: 4.62 (1.9)
					1 mo: 4.24 (2.1)
					Negative correlation of 25(OH)D with body weight (r = -0.378, p = 0.015) at baseline
					No significant differences at baseline or 1 mo in markers of bone turnover, 1,25-(OH)2D or PTH between groups with 25(OH)D < 50 nmol/L or > 50 nmol/L at baseline

BMC, bone mineral content; BMD, bone mineral density; BMAD, bone mineral apparent density; CG, control group; CPBA, competitive protein binding assay; d, day; DXA, dual X-ray absorptiometry; IG, intervention group; iPTH, intact p; arathyroid hormone; LSES, lower socioeconomic status; mo, month(s); FN, femoral neck; LS, lumbar spine; RIA, radioimmunoassay; SD, standard deviation; SPA, single photon absorptiometry USES, upper socioeconomic status; y, year

Confounders/effect modifiers. In the studies on older children, Javaid adjusted for the age of the child at the time of the BMC measurement due to the strong association between age and whole body BMC.¹⁰¹ Since bone size can affect the BMD results, volumetric BMD at the lumbar spine was calculated. For adolescents in the 25(OH)D-BMC/BMD cohort study,¹⁰³ adjustments were made for the time to followup, and regression analyses were performed to determine covariates for BMD and BMC. El-Hajj Fuleihan¹⁰⁵ made adjustments for lean mass and bone area, and did exploratory subgroup analyses on pre and post menarcheal girls in their analysis of vitamin D status in relation to BMD and BMC. Marwaha¹⁰⁶ adjusted BMD for both height and weight.

Study quality. On the Jadad scale, one RCT scored 3/5¹⁰² and one scored 4/5¹⁰⁵ indicating both were of high quality. The overall study quality for the observational studies was fair. Limitations included failure to adjust for relevant confounders or other sources of bias, and higher numbers of participants lost to followup.

Qualitative synthesis of individual study results. In a study of pre-pubertal Finnish girls, 400 IU vitamin D₂, increased serum 25(OH)D levels (measured by RIA) compared with placebo but did not impact mid-region PTH or distal radial BMC (SPA) after 13 months.¹⁰² Radial BMC was not adjusted for bone size in this study.

In the before-after study by Rajakumar,¹⁰⁰ baseline vitamin D status (measured by CPBA with deficiency defined as a serum 25(OH)D < 25 nmol/L (10 ng/ml) and insufficiency defined as ≤ 50 nmol/L) was negatively correlated with PTH (but not associated with baseline serum calcium, phosphorus, albumin, or 1,25-(OH)₂D). Serum PTH remained stable at levels of 25(OH)D around 75 nmol/L. There were no significant differences between the vitamin D insufficient and sufficient groups with regard to gender, weight, height, BMI and skin pigmentation. The mean (SD) daily dietary vitamin D intake was 277 (146) IU (mean intakes of 233 in the insufficiency group and 318 IU in the sufficient group were not significantly different). Dietary calcium intake was significantly higher in the sufficient group.

Javaid¹⁰¹ reported that low serum 25(OH)D concentrations (measured by RIA) in mothers during late pregnancy were weakly but significantly associated with reduced whole body (r = 0.21, p<0.01) and lumbar spine (r = 0.017, p = 0.03) age-adjusted BMC (DXA-Lunar DPX-L). Bone mass in children of mothers who were vitamin D deficient (25(OH)D < 28 nmol/L) during pregnancy was significantly lower compared to children born to vitamin D sufficient mothers. Reduced umbilical venous calcium also predicted reduced childhood bone mass (p = 0.0286). Whether this observation is mediated, totally or in part, through an effect on bone size and/or muscle mass is not clear. Maternal vitamin D status was positively associated with whole body and spine BMC in the offspring, and neither childhood height nor lean mass was associated with maternal 25(OH)D levels. Adjustment for childhood height did not significantly weaken the relation between maternal vitamin D status and whole body BMC. In contrast, volumetric BMD of the lumbar spine (which corrects for bone size) was not associated with maternal vitamin D status. Milk intake and physical activity at age nine were not significant determinants of bone mass although these findings do not rule out the possibility that factors such as UV exposure, diet and other lifestyle characteristics may have affected bone mass. When socioeconomic status was adjusted for, it did not change the association substantially. The type of postnatal feeding in the first three months also did not affect bone mass.

For girls age 9 – 15 years, the three year cohort study (N = 171) by Lehtonen-Veromaa evaluated the relation between baseline 25(OH)D levels (measured by RIA) and the change in lumbar spine (r = 0.35, p < 0.001) and femoral neck BMD (r = 0.32, p < 0.001). Baseline 25(OH)D also correlated with the change in LS BMAD (size-corrected form of BMD) (r = 0.35, p < 0.001) and FN BMAD (r = 0.24, p < 0.002). The difference in the percent increase from baseline in lumbar spine BMD (adjusted for the followup period) between those with low 25(OH)D levels (<20 nmol/L) and those with higher 25(OH)D levels was four percent. The difference in lumbar spine BMD was 12.7, 13.1 and 16.7 percent for the lowest, middle and highest 25(OH)D tertiles, respectively.¹⁰³

In another cohort (N = 175) of French teenage boys, there was a significant negative correlation between serum iPTH and 25(OH)D levels (measured by CPBA), with a plateau in PTH demonstrated at 25(OH)D levels of 83 nmol/L and above.¹⁰⁴ At this level of 25(OH)D, the iPTH reached a plateau at 2.48 pmol/L.

El-Hajj Fuleihan¹⁰⁵ found a significant association between baseline serum 25(OH)D levels (measured by CPBA) and baseline BMD at the lumbar spine (r=0.16, p=0.033), femoral neck (r = 0.17, p = 0.028), and radius (r = 0.24, p = 0.002) (DXA-Hologic 4500). There was also a significant association between baseline serum 25(OH)D levels and baseline radius BMC (r = 0.16, p = 0.033). The mean baseline serum 25(OH)D was 35 nmol/L (14 ng/ml). In post hoc analyses, there were negative correlations between baseline serum 25(OH)D levels and percent change in lumbar spine BMD (r = -0.16, p = 0.044) or subtotal body BMD (r = -0.20, p = 0.009) over one year. Significant negative associations were found between baseline serum 25(OH)D levels and percent change in spine, femoral neck and radius BMC.

After vitamin D supplementation for one year, total hip BMC increased in the high dose (14,000 IU/wk) group (pre- and post-menarcheal girls combined) but there were no significant changes in BMC or BMD at other skeletal sites. In an exploratory subgroup analysis in pre-menarcheal girls alone (N = 34), total body lean tissue mass increased in both supplementation groups. Lumbar spine areal BMD was significantly increased in the low dose (1,400 IU/wk) group, and trochanter BMC was increased in both the high and low dose groups. The magnitude of the treatment effect was not significant after adjusting for both bone area and lean tissue mass. The authors acknowledge a limitation of DXA in evaluating areal BMD and BMC is the lack of consensus on how best to adjust for bone size. In postmenarcheal girls, there were no differences in changes in lean mass, BMD or BMC amongst the three groups. In boys (data not shown), the authors reported there was no consistent positive effect of vitamin D supplementation on lean mass, BMD or BMC.

Marwaha¹⁰⁶ showed that children with a lower socioeconomic status had significantly lower 25(OH)D concentrations (measured by RIA) and mean BMD (unadjusted for bone size) for the forearm and calcaneus (DXA-PIXI-1.34) was higher in the upper socioeconomic group. There was a significant negative correlation between serum immunoreactive PTH and 25(OH)D concentrations (r = -0.202, p < 0.001). PTH concentrations only increased at 25(OH)D concentrations below 12.5 nmol/L. There was no significant correlation between the mean serum concentration of 25(OH)D and BMD in both groups.

Overview of Relevant Study Characteristics and Results

Table 4

Serum 25(OH)D Levels and Bone Health Outcomes in Pregnant (more...)

Table 4

Serum 25(OH)D Levels and Bone Health Outcomes in Pregnant or Lactating Women

Author (year) Country, Funding	Population, N Attrition Mean age Ethnicity	Duration	Serum 25(OH)D mean (SD) (nmol/L) Assay	Bone Health Outcomes	Results
Prospective Cohorts
Ardawi (1997)109	40 Pregnant women	6 wks	25(OH)D	iPTH (IRMA)	Serum 25(OH)D declined significantly from 1st to 3rd trimester and remained low through 6 wks postpartum. No values were < 20 nmol/L.
Saudia Arabia	280 Non-pregnant women		Pregnant women:		PTH (pregnant women):
Public	NR		1st trimester: 54 (10)		Serum 25(OH)D levels correlated negatively with serum iPTH (r=-0.62, p <0.001);
	NR		2nd trimester: NR		1st trimester: 1.31 (0.25)
	Pregnant women 26.8 (5.8) y;		3rd trimester: 33 (8) term: 35 (11)		2nd trimester: 2.26 (0.39) term: 1.86 (0.87);
	non-pregnant women 27.8 (5.3) y		6 wks postpartum: 33 (8)		6 wks postpartum: significant increase compared to pregnancy values (~ 3.5, graph only, exact value NR)
	Arab		CPBA		Serum 25(OH)D in pregnancy correlated positively with 1,25-(OH)2D (r=0.52, p < 0.001), serum PTH-related peptide (r = 0.51, p < 0.001), serum Ca (r=0.23, p < 0.001), serum Mg (r=0.62, p < 0.01)
Morley (2006)110	475 Pregnant women recruited at < 16 wks gestation from antenatal clinic	NA	25(OH)D geometric mean at recruitment:	PTH (chemiluminescent enzyme-labelled immunometric assay)	After adjustment for seasonal variation, increase in 25(OH)D concentrations between early and late pregnancy: gemometric mean ratio 1.06, 95% CI 1.02, 1.10, p = 0.004
Australia	Unclear if recruitment was consecutive		In summer: 62.6	Infant linear growth (head, mid-arm, calf circumference) Knee-heel length	No association between maternal 25(OH)D and PTH levels at recruitment (11 wks gestation)
Public	21% attrition		In winter: 49.2, p < 0.001		Positive association between maternal PTH and measures of infant size (to knee-heel length, birth weight) independent of 25(OH)D status.
	29.3 (6.4) y		% < 28 nmol/L:		Mothers with serum 25(OH)D < 28 nmol/L, at 28–32 wk gestation, had babies with: shorter (-0.7 wk) gestation length, and knee heel length (-2.7mm) after adjustment for gestation length, and lower birth weight (-157 g) than those with 25(OH)D ≥28 nmol/L
	98.6% Caucasian (excluded those thought to be at high risk for deficiency including dark skinned individuals) 105 White, 7 Asian American, 3 African American		In summer: 0.8%
			In winter: 9.4%, p < 0.001
			At 28 – 32 wks gestation:
			In summer: 48.3
			In winter: 68.9, p < 0.001
			% < 28 nmol/L
			In summer: 3.7%
			In winter: 10.0%, p = 0.02
			RIA
Sowers (1998)108	115 Women in third trimester, with a parity of 0 – 1, recruited on basis of intent to breast-feed or formula-feed exclusively.	18 mo	25(OH)D	BMD: FN and LS (DXA-Lunar)	25(OH)D concentration was not predictive of changes in FN or LS BMD or bone turnover markers.
U.S.	2 wks: N = 115; 18 mos: N = 71		postpartum stages:	PTH (midmolecule, RIA)	Pattern of decline in 25 (OH)D concentration over 18 mo period was independent of lactation status
Public	Mean age: 29.3 (20–40) y		2 wks 40.3 (11.3)		PTH, 25(OH)D and 1,25-(OH)2D had no association with prolactin or PTH-related peptide and did not differ by lactation practice.
	91% Caucasian; 6% Asian American; 3% African American		2 mo 30.1 (7.5)
			4 mo 37.4(10.5)
			6 mo 33.6 (10.4)
			12 mo 29.5 (8.4)
			18 mo 27.0 (7.3)
			RIA
Before-After Studies
Datta (2002)111	160 Consecutive ethnic minority pregnant women in the U.K. recruited at first antenatal visit; those identified as vit D def (serum 25(OH)D < 20 nmol/L) were treated with vit D 800 IU/d and followed to delivery	Early pregnancy to delivery	25(OH)D	PTH levels provided for vit D def women only	At baseline, 65 of 80 (81%) women with serum 25(OH)D < 20 nmol/L had normal PTH (< 5.6 pmol/L)
Wales	Attrition: 58/80 (73%) vit D def women had post treatment (post delivery) assessment		80/160 (50%) had 25(OH)D < 20 nmol/L		35/58 (60%) re-tested at delivery had 25(OH)D within normal range
Funding NR	Mean age NR		Reported for vit D def women only:		At delivery, mean serum 25(OH)D increased from 15 to 27.5 nmol/L, but mean PTH level remained the same
	African (N = 36), Afro-Caribbean (N = 4), Indian (N = 100), Middle Eastern (N = 9), Far Eastern (N = 11)		Recruitment: 14.5 (2.3)		serum PTH mean (SD) pmol/L: at recruitment: 3.69 (2.78) pmol/L end of study (post treatment): 4.06 (3.17), NS
			End of study (with treatment): 28.1(15.9)		Compliance with vit D not measured
			Vit D status at delivery in those treated with supplements reported for 58/80
			RIA

total 25(OH)D or either isoform of 25(OH)D (isoform not specified);

def, deficient or deficiency; IRMA, immunoradiometric assay; IU, international units; Mg, magnesium; NR, not reported; PTH, parathyroid hormone; RIA, radioimmunoassay; SD, standard deviation; vit, vitamin; wk. weeks; y, year;

The time of assessment of vitamin D status, the assay method for 25(OH)D and bone health outcomes varied across studies which precluded quantitative synthesis of results.

Qualitative Synthesis of Individual Study Results

Maternal vitamin D status. In the study of non-European minority women from South Wales,¹¹¹ 50 percent of the women were vitamin D deficient at the first antenatal visit, using a criterion of serum 25(OH)D < 20 nmol/L. Vitamin D supplementation (800–1600 IU) D during pregnancy normalized vitamin D status in 60 percent of the deficient group. In the study in Saudi Arabia of 40 Asian women,¹⁰⁹ serum 25(OH)D declined significantly from baseline (about 11 weeks gestation) to the third trimester (mean of 31.4 wk of gestation) and remained low through to 6 weeks post-delivery. However, at all timepoints, mean serum 25(OH)D concentrations were within the normal range of a reference group of non-pregnant women (N = 280) who were healthy and non-lactating, suggesting that although serum levels decline during the end of the third trimester, they do not differ extensively from those of the non-pregnant state. None of the pregnant women were classified as having subclinical vitamin D deficiency (25(OH)D < 20 nmol/L). In the study¹¹⁰ in primarily Caucasian women in Australia, serum 25(OH)D was similar at recruitment (11 weeks of gestation) and at the beginning of the third trimester of pregnancy (28–32 weeks of gestation) but there were significant differences between mean values in winter versus summer months. The percent who were vitamin D deficient (9–10 percent as defined by 25(OH)D < 28 nmol/L) was significantly greater in winter than summer.

One cohort study assessed vitamin D status postpartum and in relation to breast-feeding.¹⁰⁸ There was a non-significant trend to a decline in vitamin D status in the initial 2–4 months and the pattern was not influenced by the season of birth. Vitamin D status was not influenced by the duration of breast-feeding. The percent of women who were vitamin D deficient was not provided but based on the mean values, some of the women would have had 25(OH)D values less than 20 nmol/L. Data on vitamin D intake or sun exposure were not provided.

Vitamin D status and bone health outcomes. In the cohort study by Sowers, bone mineral density of lumbar spine and femoral neck was measured in 115 mothers with different breast-feeding practices during the postpartum period and vitamin D status was not associated with changes in BMD of the femur or spine.¹⁰⁸ Women were recruited during the third trimester, lumbar spine BMD was measured at two weeks, 6, 12 and 18 months postpartum and femoral neck at two weeks, two, four, six, 12 and 18 months. Serum PTH and the other calciotrophic hormones were not associated with changes in femoral or lumbar spine BMD, suggesting that 25(OH)D, PTH and 1,25-(OH)₂D do not explain the calcium mobilization and bone turnover that occurs during lactation.¹⁰⁸

In the before-after study in pregnancy,¹¹¹ serum 25(OH)D did not appear to correlate with serum PTH concentrations, with 65/80 women with low 25(OH)D having PTH in the normal range.

In a prospective cohort study on 40 Asian women (280 non-pregnant controls),¹⁰⁹ serum 25(OH)D levels negatively correlated with intact PTH (r = -0.62, 0<0.001). In this study, serum osteocalcin, a bone formation marker was below the reference range observed in non-pregnant women, and declined in the second trimester compared to the first, but then rose to within or above the reference range at term and 6 weeks postpartum. This suggests changes in bone turnover do occur during early pregnancy, irrespective of normal vitamin D status.

In the prospective cohort study by Morley there was no association between baseline maternal 25(OH)D concentrations and measures of infant size at birth.¹¹¹ There was an inverse association between maternal log₂ 25(OH)D and log₂ PTH. Using the maternal 25(OH)D concentrations at 28–32 weeks, the mean gestational length was significantly shorter (0.7 weeks, 95% CI -1.3,-0.1 weeks) in the vitamin D-deficient mothers compared to mothers with 25(OH)D concentrations over 28 nmol/L. This association was not altered by inclusion of log₂ PTH, serum calcium and albumin concentrations. Infants born to mothers who were vitamin D deficient at 28–32 weeks gestation, had lower mean knee-heel length (-2.7 mm) compared to infants born to mothers who were not vitamin D deficient, after adjusting for gestation length.¹¹⁰ Further non-parametric smooth regression analysis and adjustment of confounders suggested the possibility of a linear association when 25(OH)D levels were below 30–40 nmol/L, but there was no association at higher 25(OH)D levels. Low maternal 25(OH)D levels were associated with a negative impact on long bone growth and the authors postulated that maternal PTH may affect fetal growth via an affect on 1,25-(OH)₂D production.¹¹⁰

Study quality. There were no RCTs identified that evaluated the association between serum 25(OH)D concentrations and bone health outcomes in pregnant and lactating women. The before-after study¹¹¹ was poorly designed, lacked detail regarding the duration and compliance with the vitamin D supplements, and the analyses were incomplete. A limitation of the included studies was failure to adjust for all relevant covariates. Only one six-week cohort study was considered to be of good quality, since it included an age-matched non-pregnant cohort with control values for all biochemical measurements (N = 280) and provided six serial measures with no attrition during followup.¹⁰⁹ The cohort study conducted during lactation,¹⁰⁸ was of good quality as it included six serial biochemical measures, four measures of spinal BMD and six of femoral neck BMD throughout lactation, and adjusted for a number of covariates. The one study in which the primary outcome was size of offspring at birth was judged to be of fair quality due to loss of followup of over 20 percent.¹¹⁰

Overview of Relevant Studies

Table 5

Studies Reporting Serum 25(OH)D Levels and Bone Health (more...)

Table 5

Studies Reporting Serum 25(OH)D Levels and Bone Health Outcomes in Postmenopausal Women and Older Men

Outcome (N studies)	Study Design	Associations
Fractures (N=15)	RCTs=0	Association:
	Cohorts=3	1 cohort131
	Case-controls=12	9 case-controls29,139,141,142,144,146,148,150,151
		No Association:
		2 cohorts130,133
		3 case-controls137,145,149
Falls (N=5)	RCTs=1	Association:
	Cohorts=3	1 RCT114
	Case-controls=1	1 cohort123
		1 case-control138
		No Association:
		2 cohorts122,134
BMD/BMC (N=19)	RCTs=6	Association:
	Cohorts=7	1 RCT119
	Case-controls=6	4 cohorts: FN BMD126,128,129,,132; 1 cohort LS BMD135
		6 case-controls: FN BMC136; FN, Tr and TH BMD139,141
		LS BMD140,143,152
		No Association:
		5 RCTs116–118,120,121
		3 cohorts: FN BMD135; proximal femur, LS BMD127; FN, LS BMD131
Performance measures (N=7)	RCTs=3	Association:
	Cohorts=4	2 cohorts124,131
		2 RCTs113,115
		No Association:
		2 cohorts125,134
		1 RCT112

BMC, bone mineral content; BMD, bone mineral density; FN, femoral neck; LS, lumbar spine; RCTs, randomized controlled trials; TH, total hip; Tr, trochanter

Table 6

Serum 25(OH)D Levels and Fractures in Postmenopausal (more...)

Table 6

Serum 25(OH)D Levels and Fractures in Postmenopausal Women and Older Men

Author (year) Country Funding	Population, N Gender Mean age (SD) Ethnicity	Matching Variables	Duration	25(OH)D Mean (SD) nmol/L Assay	Bone Health Outcomes	Covariates Summary of Results
Prospective Cohorts
Cummings (2006)133	Subset of a cohort of 9704 ambulatory community-dwelling women ≥ 65 years of age (nested case-control study)		5.9 y	25(OH)D	Hip fractures	Adjusted for age, weight and calcaneal BMD (SPA)
US	Groups analyzed:			22% in the subset had serum 25(OH)D ≤47.5 nmol/L	vertebral fractures	There were no statistically significant unadjusted or adjusted (age, weight, season, use of vit D supplements) association between serum 25(OH)D or PTH and the risk of hip or vertebral fracture.
Public	Of the 332 women in the cohort who had hip fractures, 133 were randomly selected;			RIA	BMD calcaneus (SPA)	For women in the lowest quintile of serum 25(OH)D levels, there was no increased risk for hip or vertebral fracture.
	Of the 389 women who had new vertebral fractures in the cohort, 138 were randomly selected; 359 ctrls were randomly selected; of these, 343 served as ctrls for hip fracture cases and 264 served as ctrls for vertebral fractures (based on availability of XRs)				PTH (measured by IRMA)	Women in the lowest quintile of serum 1,25-(OH)2D had a significant increase in hip fracture risk (RR 2.1, 95% CI 1.2–3.5) but not vertebral fracture risk.
	100% female
	72.6 y (subset)
	White
Gerdhem (2005)131	1,044 Ambulatory independently living women		3 y	25(OH)D	Fractures (low energy)	119/986 (12%) had a total of 159 low energy fractures (29 hip, 28 wrist, 12 proximal humerus, 43 vertebral and 47 other)
Sweden	58/1044 (6%) did not complete			95 (30)		9/43 (21%) with 25(OH)D < 50 nmol/L had one or more fractures vs. 110/943 (12%) with 25(OH)D > 50 nmol/L: HR 2.04 (95% CI, 1.04 – 4.04).
Public	100% female			< 50 nmol/L: 4.4%		Fracture association was independent of season although a seasonal difference was noted in mean level of 25(OH)D (Sept 101 nmol/L vs. Feb 89.8 nmol/L).
	75 y (range 75–75.9 y)			< 75 nmol/L: 26%
	NR			CPBA
Woo (1990)130	427 Elderly ≥ 60 y living independently in sheltered housing.		30 mo	25(OH)D	Fractures	Adjusted for age, gender, drinking, smoking and BMI.
Hong Kong	144/427 (34%)			fracture subset (N=9) 63.3 (6.9) vs. no fracture subset 74 (1.15), NS		Subjects with lower serum 25(OH)D (males < 79 nmol/L and females < 66 nmol/L) had a nonsignificant increase in adjusted RR for fracture.
NR	60% females			CPBA
	Women: 70 y
	Men: 69 y
	Asian (Chinese)
Case-Control Studies
Bakhtiyarova (2006)151	64 Hip fracture cases (spontaneous or low trauma)	NR		25(OH)D	Hip fractures	Median serum 25(OH)D levels significantly lower in hip fracture cases vs. ctrls (graph only).
Russia	97 ctrls admitted to opthamology dept			Cases: 22.4 (11.4)		Hip fracture patients more likely to have serum 25(OH)D < 25 nmol/L than ctrls (65% vs. 47%, p=0.006).
NR	Cases: 69% female Ctrls: 55% female			Ctrls: 28.1 (10.1)
	Cases: 68.8 (9.5) y Ctrls: 70.2 (8.3) y			25(OH)D <25 nmol/L:
	White (Caucasion)			Cases: 65%; Ctrls: 47%
				25(OH)D<40 nmol/L:
				Cases 89%; Ctrls 89%;
				25(OH)D <50 nmol/L:
				Cases 100%, Control 98%
				CPBA
Boonen (1997)142	117 Elderly women with hip fractures and 117 community-dwelling ctrls	Age, PM status, gender, ethnicity		25(OH)D	Hip fractures	Serum 25(OH)D significantly lower in cases vs. ctrls (p=0.001).
Belgium	100% female			Cases 25.25 (22)	BMD (FN and Tr) (DXA)	Hip BMD (FN and Tr) significantly lower in cases vs. ctrls (p < 0.001).
Public	Cases: 79.2 y			Ctrls: 53.75 (33.25)
	Ctrls: 77.7 y			CPBA
	White (Caucasion)
Boonen, (1999)139	100 Postmenopausal women	Age, gender, PM status, sampled at the same time of year		25(OH)D3	Fractures	Adjusted for age
Belgium	50 osteoporotic hip fracture patients and 50 independently living ctrls			Cases: 29.3 (26.5)	BMD (FN and Tr) (DXA)	Mean 25(OH)D3 was significantly lower cases vs. ctrls.
Public	100% female			Ctrls: 68.75 (39), p < 0.001	PTH (IRMA)	25(OH)D < 30 nmol/L:
	Cases: 74.2 (7.8) y			CPBA		64% of cases vs. 8% ctrls within the same
	Ctrls: 75.8 (5.6) y					4 mo sampling period (no relation b/w
	NR					25(OH)D and mo of sample collection).
						FN and Tr BMD were significantly lower in cases than ctrls.
						No significant relation b/w the 25(OH)D3-PTH axis and BMD when analyzed separately. In multiple regression analyses of pooled data, models using 25(OH)D3 and PTH were predictive of FN BMD (R2=32%, p<0.001).
Cooper (1989)145	41 Hip fractures	Age (cases and one of the two control groups similar), gender		25(OH)D	Hip fractures	Age and albumin
UK	40 Healthy ctrls (20 inpatient and 20 outpatient)			Fracture patients: 23.5 (14.5),	PTH (immunoreactive, C-terminal)	Mean 25(OH)D was significantly lower in cases vs. ctrls (p<0.01). When age and albumin were used as covariates in the analysis, there was no residual difference in serum 25(OH)D levels.
NR	100% female			Inpatient ctrls: 35.75 (23.5)		More hip fracture cases (49%) had 25(OH)D levels <25 nmol/L vs. 15% of inpatient and 10% of outpatient ctrls.
	Cases 77.4 (8.6) y			Outpatient ctrls: 48.5 (25)
	Ctrls 73.3 (10.5) (inpatients), and 66.9 (11.8) y (outpatients)			25(OH)D <20 nmol/L):
	NR			Cases: 49% vs.
				Ctrls: 10 – 15%
				RIA
Diamond (1998)150	41 Men with hip fracture	Age, gender		25(OH)D	Hip fractures	Age, body weight, comorbidity score, smoking history, alcohol intake, serum calcium, albumin, 25(OH)D and free testosterone.
Australia	82 healthy ctrls (41 inpatient and 41 outpatient)			Cases 45.6, range 36.9–52.3		Men with hip fractures had significantly lower 25(OH)D levels vs. ctrls (p=0.007). 25(OH) D < 50 nmol/L:
NR	100% male			Inpatients ctrls 61.1 (range 50.0–72.2)		63% of fracture patients vs. 25% of combined ctrls, OR 3.9 (95% CI 1.74 –8.78).
	Cases: 79.6 y			Outpatients ctrls 65.9 (range 59.0–72.8), p = 0.007 for cases vs. combined ctrls		Multiple regression analysis showed that serum 25(OH)D level < 50 nmol/L was strongest predictor of hip fracture (r = 0.34 (0.19), p=0.013).
	Ctrls: 78.7 y and 77 y			RIA		Age was the best determinant of a serum 25(OH)D level < 50 nmol/L, p=0.028
	NR
Erem (2002)137	21 Women with hip fractures and 20 healthy PM women, all independent community-dwellers	Age, gender, PM status		25(OH)D	Hip fractures	NR
Turkey	100% female			Cases 26.9 (25.0)		Non significant difference in 25(OH)D levels in hip fracture patients vs. ctrls
Public	Cases: 76.7 (6.5) y Ctrls: 75.4 (6.3) y			Ctrls: 24.9 (20.5)		25 (OH)D levels in all groups < 37.5 nmol/L
	Far Eastern			CPBA
Landin-Wilhelmsen, (1999)140	128 PM women with osteoporosis	Age, gender, PM status		25(OH)D3	Fractures	NR
Sweden	227ctrls from outpatient clinic			Cases: 88 (30)	BMD and BMC: LS, TB and FN (DXA)	25(OH)D significantly lower in osteoporotic women vs. ctrls (p<0.05); PTH significantly higher in osteoporotic women vs. ctrls (p < 0.001)
Public	100% female			Ctrls: 96 (32)	PTH (IRMA)	Fracture history in 56% of osteoporotic women vs. 4% of ctrls, p<0.001
	osteoporotic women: 59 (6) y			RIA		osteoporotic women had lower body weight and BMI vs. ctrls (p<0.001).
	ctrls: 59 (5) y
	NR
Lau, (1989)144	200 hip fracture patients in hospital and 427 community-living ctrls	Ethnicity		25(OH)D	Hip fractures	NR
Hong Kong	NR			Men		25(OH)D levels were significantly lower in cases vs. ctrls (p<0.01).
NR	Age range: 49–93 y (cases), 60–90 y (ctrls)			cases <70 y: 56.3 (18) and ≥70 y:		Hip fracture patients with low 25(OH)D (male < 36.5 nmol/L, female, < 34.3 nmol/L, defined by lower limit of 95% CI for ctrls) were less mobile than those with normal 25(OH)D; 33% with low 25(OH)D could walk outdoors without an aid vs. 61% of those with a normal 25(OH)D level.
	Asian			46.3 (17.3)
				Ctrls <70 y: 84.8 (25.5) and ≥70 y:
				80.5 (21.5
				Women
				cases <70 y: 44.5 (13.8) and ≥70 y:
				42.8 (15.5)
				ctrls <70 y: 72.5 (15.5) and ≥70 y:
				65 (17)
				CPBA
LeBoff (1999)29	98 community-dwelling women	Gender, PM status, setting, surgical procedure		25(OH)D	Hip fractures	Adjusted for age and estrogen replacement therapy.
U.S.	30 with hip fracture and osteoporosis (OP) (group 1);	OP in group 1 and subset of group 2		median:	BMD: LS, FN, Tr, total body (DXA)	Women with hip fracture and OP had significantly lower 25(OH)D vs. women with OP admitted for surgery (p=0.01) and vs. women without OP admitted for surgery (p=0.02).
Public	68 women admitted for elective joint replacement with (17) or without (51) osteoporosis (group 2)			Group 1: 32.4,		% of women with 25(OH)D < 30 nmol/L: Signficantly more in group 1 (50%) vs. OP or non-OP group 2 (graph only ~ 5% for OP and 10% for non-OP) (p < 0.002).
	100% female			Group 2: OP 49.9;		Mean BMD (LS, FN, Tr) was significantly less in women with acute hip fracture/OP vs. elective surgery non-OP ctrls.
	Group 1: 77.9 y			non-OP 55.0
	Group 2: OP 69.9 y; non-OP 64.4 y			RIA
	NR
Lips (1983)147 and Lips (1987)146	125 consecutive patients with femoral neck fracture and 74 healthy community ctrls	Age		25(OH)D	Hip fractures	Adjusted for age and sex
The Netherlands	Cases: 67% female			Cases: 18.5 (10.6) Ctrls: 32.9 (13.6)		Serum 25(OH)D levels lower in cases vs. ctrls (p<0.001).
Public	Ctrls: 73% female			serum 25(OH)D < 20 nmol/L:
	Cases: 75.9 (11) y			Cases: 62%
	Ctrls: 75.6 (4.2) y			Ctrls: 16%
	NR			CPBA
Lund (1975)149	67 consecutive cases of proximal femur fractures ctrls: milddle aged (30–59 y) N = 27 and elderly healthy individuals (60–95 y) N = 67 at same time of year	Age		25(OH)D	Proximal femur fractures	There was no statistically significant difference in serum 25(OH)D levels vs. either ctrl.
Denmark	NR			range 7.5–195 nmol/L
NR	NR			N=12 (18%) <25 nmol/L
	NR			CPBA
Punnonen (1986)148	40 cases of hip fracture and 25 ctrls (from gynecological clinic)	Age, gender, setting		25(OH)D	Hip Fractures (FN)	NR
Finland	100% female			Cases: 18.2 (13.2)		25(OH)D levels were significantly lower in cases vs. ctrls, (p<0.01).
NR	Cases: 77.1 (8.6) y			Ctrls: 53.3 (24.1)
	Ctrls: 73.8 (8.4) y			CPBA
	NR
Thiebaud, (1997)141	179 Hip fracture patients;	Age, setting (for cases and one control group)		25(OH)D	Fractures	Adjusted for age, sex, and creatinine
Switzerland	180 hospital ctrls; 55 community ctrls			Women:	BMD: FN, TH and Tr (DXA)	Women and men with hip fractures had significantly lower 25(OH)D levels vs. ctrls. Fracture patients had lower hip (TH, FN) BMD vs. either ctrl group (p < 0.001).
Public	Cases: 76% female			Fracture cases:		In multivariate logistic regression of the risk for hip fracture, serum albumin and PTH were significant. In women, BMD was weakly correlated with 25(OH)D and the only significant association was at the Tr (r=0.13, p < 0.05).
	Hospital Ctrls: 75% female			25.5 (20.5)
	Community ctrls: 85% female			Hospital ctrls:
	Cases: women 81.0 y; men 77.7 y			31.5 (26.5)
	Hospital ctrls: women 80.9, men 76.9 y			Community ctrls:
	Community ctrls: women 71.7 y, men 71.3 y			53 (23)
				Men
				Fracture cases:
				17.25(18.5)
				Hospital ctrls:
				27.75 (21.5)
				Community ctrls:
				31.5(22.8)
				RIA

Note:

total 25(OH)D or either isoform of 25(OH)D (isoform not specified);

BMC, bone mineral content; BMD, bone mineral density; ctrls, controls; DXA, dual energy X-ray absorptiometry; FN, femoral neck; PM, post menopausal; RIA, radioimmunoassay; SD, standard deviation; SPA, single-photon absorptiometry; TH, total hip; Tr, trochanter; wks, weeks; y years

Table 7

Serum 25(OH)D Levels and Falls and/or Performance Measures (more...)

Table 7

Serum 25(OH)D Levels and Falls and/or Performance Measures in Postmenopausal Women and Older Men

Author (year) Country Funding	Population, N Attrition Gender Mean age (SD) Ethnicity	Intervention Duration	Serum 25(OH)D Mean (SD) nmol/L Assay	Bone Health Outcomes	Covariates Summary of Results	Jadad AC
RCTs
Bischoff-Ferrari (2003)114	122 Elderly women in long-stay geriatric care	IG: 800 IU D3	25(OH)D	Falls	Age, height, weight, BMI, number of falls in pre-treatment period, being a faller in the pre-treatment period, prior vit D use, comorbidity index. Muscle strength, use of walking aid, baseline 1,25-(OH)2D, 25(OH)D, iPTH, albumin and observation time during treatment	3
Switzerland	drop outs	+1200 mg Calcium carbonate daily	Median (IQR):	iPTH (RIA)	Vit D + Ca accounted for 49% reduction in falls (-0.68; 95% CI 14–71%, p=0.01) after adjustment for age, number of falls in pretreatment period, being a faller in pre-treatment period, baseline 1,25-(OH)2D, and 25(OH)D. Predictors other than treatment were being a faller, number of falls in pre-treatment period and age.	Unclear
Public and private	IG1: 31%	CG: 1200 mg	baseline
	CG: 25%	Ca daily	IG1: 30.75 (23–55)
	100% female	12 wks (6 wk pre-treatment)	CG: 29 (23–55)
	85.3 y		values < 30
	range 63–99		nmo/L: 50%.
	NR		End of study
			IG1: 65.5 (49.75–82.75)
			CG: 28.5 (24.5–41.5)
			RIA
Corless (1985)112	82 Elderly hospital patients with serum	IG1: 9,000	25(OH)D	ADLs: muscle strength and independence index	NR	5
U.K.	25(OH)D < 40 nmol/L	IU/d D2	Mean (SEM):		No significant correlation between change in 25(OH)D and change in ‘muscle strength’ (r=0.12, p>0.3) or ‘independence’ indices (r=0.26, p>0.1).	Unclear
Public	Drop outs	CG: placebo	Baseline
	IG1: 9/41 (22.1%),	9 mo	IG1: 16.6 (2.1)
	CG: 8/41 (19.5%)		CG: 17.6 (2.05)
	IG1: 78.1% female		% < 20 nmol/L:
	CG: 78.8 % female		IG1: 66%
	IG1: 82.3 (6.0) y		CG: 70%
	CG: 82.6 (6.9) y		End of study:
	NR		graph only (IG1: ~ 110 nmol/L)
			CPBA
Dhesi, (2004)115	139 Ambulatory older adults with a history of falls and 25(OH)D <30 nmol/L	IG1: 600,000,	25(OH)D	Falls, postural sway, reaction time, aggregate functional performance time and quadriceps strength	NR	5
U.K	Drop outs	D2 (injection)	Baseline		Significant correlation between Δ 25(OH)D and Δ aggregate functional performance time in both groups (r=0.19, p=0.03).	Unclear
Public	IG1: 8/70 (11.4%),	CG: placebo	IG1: 26.8 (25.5–28)
	CG: 8/69 (11.6%)	6 mo	CG: 25 (23.8–26.3)
	IG1: 75.7% female		End of study
	CG: 79.7% female		IG1: 43.8 (41.3–46.3)
	IG1: 77.0 (6.3) y		CG: 31.5 (28.5–34.5)
	CG: 76.6 (6.1) y		RIA
	Caucasion
Kenny (2003)113	65 Healthy, community-dwelling men with normal 25(OH)D	IG1: 1,000 IU	25(OH)D	Ability to rise from a chair, static balance, 8-foot walk, TUG, timed supine to stand test and PASE questionnaire.	NR	4
U.S.	IG1: 4/33 (12.1%),	D3 + 500 mg Ca	Baseline		Association between baseline 25(OH)D and single-leg stance time (r=0.245, p<0.05) and PASE Score (r=0.360, p<0.01).	Adequate
Public	CG 1/32 (3.1%)	CG: 500 mg Ca daily	IG1: 65 (17.5)
	100% male	6 mo	CG: 60 (17.5)
	IG1: 77 y		End-of-study (graph only)
	CG: 75 y		IG1: ~ 83
	NR		CG: ~ 50
			CPBA
Prospective Cohorts
Faulkner (2006)134	9,704 Older community-dwelling women (from the Study of Osteoporotic Fractures), and 389/400 (97.2%) drawn at random from entire cohort for serum measures	4 y	25(OH)D3	Falls; GS, quadriceps strength, chair-stand time, walking speed, reaction time and balance-walk time measured in subset of 389	Adjusted for age, height, BMI, clinical site, season of serum collection, education, ethnicity, physical activity, smoking, alcohol use, housebound status, dietary calcium intake, orthostatic hypotension, stroke, Parkinson's disease, arthritis, diabetes, osteoporosis, hyperthyroidism, cognitive impairment, visual acuity, self-rated health, use of estrogen, thyroid hormones, calcium supplements, corticosteroids, diuretics, and CNS-active medications.
U.S.	100% female		Median (IQR)		There was a trend toward higher 25(OH)D3 concentrations associated with weaker grip strength (p=0.017) vs. women in the first quartile.
Public	Median (IQR):		Total cohort: 62.5 (47.5–77.5) Women using vit D supplements (N=4,273): 67.5 (52.5 – 85)		25(OH)D3 was not associated with neuromuscular function, Δ neuromuscular function (grip strength, chair stand time, walking speed and balance walk time) or fall rates.
	70 (67–75) y		Women not using vit D supplements (N=5,253): 55 (42.5–70)
	66% Northern European (excluded African Americans)		% < 25 nmol/L
			Women using vit D supplements: 0.6%
			Women not using vit D supplements: 4.2%
			RIA
Flicker (2003)123	1,619 Institutionalized elderly, both low (N=667) and high level care (N=952)	145 d (low level care) and 168 d (high level care subjects)	25(OH)D	Falls	Adjusted for weight, cognitive status, psychotropic drug use, prior wrist fracture and presence of wandering behavior
Australia	All 1,619 included in analysis	3 y	Low level care:		After excluding bed bound residents and adjusting for above covariates, log serum 25(OH)D level was independently associated with time to first fall: adjusted HR 0.74 (95% CI, 0.59–0.94, p=0.01).
Public	100% female		WA (32°S): 39.3 (20.1)		20% reduction in risk of falling with doubling of 25(OH) D level.
	Low level care: 83.7 (8.7) y		NSW (34°S): 43.7 (22.5)
	High level care: 83.7 (9.1) y		Victoria (38°S) 38.4 (19.6) p<0.05
	NR		High level care:
			WA (32°S): 33 (17.3)
			NSW (34°S): 32.4 (22.4)
			Victoria (38°S): 30.7 (19.4)
			% < 25 nmol/L:
			Low level care: 22%
			High level care: 45%
			RIA
Gerdhem (2005)131	1,044 Ambulatory independently living women	3 y	25(OH)D	Gait speed, Romberg balance test, lower extremity strength	NR
Sweden	58/1,044 (6%) did not complete		95 (30)		25(OH)D correlated with: gait speed (r=0.17, p<0.001), Romberg balance test (r=0.14, p<0.001), self-estimated activity level (r=0.15, p<0.001), thigh muscle strength (r=0.08, p=0.02).
Public	100% female		< 50 nmol/L: 4.4%		5% of the variability in 25(OH)D explained by fall-related and anthropometric variables (multiple regression).
	75 (75–75.9) y		< 75 nmol/L: 26%
	NR		CPBA
Sambrook (2004)122	646 Ambulatory residents of institutional care facilities (hostels and nursing homes) > 65 y	1 y	25(OH)D	Falls	Adjusted for age, incontinence, illness severity; Interactions between PTH, 25(OH)D and other variables were tested.
Australia	9/646 (1%) did not complete		Fallers: 28.8 (14.2)		After adjusting for age, incontinence and illness severity, serum 25(OH)D was no longer a significant predictor of falls.
NR	Fallers: 84% female		Non-fallers: 33.2 (16.5)		25(OH)D was related to balance. There was a 1.65X increased risk of falls in group with 25(OH)D < 39 nmol/L and PTH > 66 pg/mL compared to those with 25(OH)D > 39 nmol/L and PTH < 66 pg/mL.
	Non-fallers: 79% female		% <39 nmol/L: 73.6%
	Fallers: 86.6 y (6.5) y		Men: 64.5%, Women: 75.8%
	Non-fallers: 85.1 (6.4) y		RIA
	NR
Visser (2003)124	1,509 Older individuals from longitudinal study of aging 501/1509 (33%) did not complete	3 y	25(OH)D	GS and ASMM Sarcopenia defined as a loss of GS > 40%, and ASSM > 3%	Adjusted for sex, age, BMI, physical activity level, chronic disease, creatinine, season of data collection and smoking.
The Netherlands	NR		NR		Separate analysis adjusted for weight change. Interactions explored between PTH and 25(OH)D
Public	Stable GS: 74.2 (6.1) y		< 25 nmol/L: 9.6% <12.5 nmol/L: 1.3%		Individuals with 25(OH)D <25 nmol/L vs. levels >50 nmol/L were more likely to experience loss of GS (adjusted OR 2.57, 95% CI 1.40–4.70, p<0.05); loss of ASMM, NS.
	Loss of GS: 76.9 y (6.5)		CPBA
	Stable ASMM: 73.7 (5.9) y
	Loss of ASMM: 74.9 (6.4) y
	NR
Verreault (2002)125	1,002 Elderly women, ≥ 65 y with moderate to severe disability living in community	3 y	25(OH)D	Lower extremity strength, GS, walking speed, repeated chair stands. Disability in activities involving mobility and upper extremity function.	Adjusted for: baseline performance, age, BMI, comorbidity and other confounders associated with a decline in performance. (Cox proportional hazard model) age, race, education, smoking and baseline BMI, season and presence of comorbidity.
U.S.	374/1002 (37%)		Mean: 52.9 % <25 nmol/L: 12.4%		No association between low 25(OH) D levels and loss of muscle strength or declines in mobility or disability. Results were similar when 25(OH)D and PTH were both included in the model.
Public	100% female		RIA
	NR
	NR

Author (year) Country Funding	Population, N Gender Mean age (SD) Ethnicity	Matching Variables	Serum 25(OH)D Mean (SD) nmol/L Assay	Bone Health Outcomes	Covariates Summary of Results	Jadad AC
Case-Control Studies
Stein (1999)138	83 ambulatory nursing home and hostel residents grouped as fallers 33) vs. never fell (50)	Age, setting, level of independence	25(OH)D	Falls	Adjusted for PTH; interactions sought between weight and gender
Australia	66% female		Median:		Serum 25(OH)D lower in patients who had a fall vs. those who did not (95% CI for difference in medians: 1 – 13 nmol/L, p=0.019).
Public	Median age (IQR): 84 (79–89) y		Cases: 22		Bivariate OR (95% CI) for falling vs. never falling for Ln 25(OH)D was 0.33 (0.13–0.83).
	NR		Ctrls: 29		Neither Ln 25(OH)D or 1,25-(OH)2D were independent predictors after adjusting for PTH.
			CPBA

AC, allocation concealment; ADLs, activities of daily living; ASMM, appendicular skeletal muscle mass; BMI, body mass index; CPBA, competitive protein binding assay; CI, confidence interval; ctrls, controls; GS, grip strength; IQR, interquartile range; NS, not significant; OR, odds ratio; PTH, parathyroid hormone; RIA, radioimmunoassay; SD, standard deviation; y, years

Table 8

Serum 25(OH)D Levels and BMD/BMC in Postmenopausal (more...)

Table 8

Serum 25(OH)D Levels and BMD/BMC in Postmenopausal Women and Older Men

Author (year) Country Funding	Population, N Attrition Gender Mean age (SD) Ethnicity	Matching Variables	Intervention Duration	Serum 25(OH)D Mean (SD) nmol/L Assay	Bone Health Outcomes	Covariates Summary of Results	Jadad AC
RCTs
Aloia (2005)117	208 Post menopausal women		IG: 800 IU D3 for 2 y, then 2,000 IU for 1 y + 1200 – 1500 mg Ca CG: 1200 – 1500 mg Ca	25(OH)D	BMD: LS, total hip, total body, mid radius (DXA)	NR	5
U.S.	IG1: 3/104 (2.9%), CG: 3/104 (2.9%) did not complete		3 y	Baseline:	PTH (IA, Allegra)	No association between serum 25(OH)D and Δ BMD. Analyses examining those with low baseline 25(OH)D or high PTH showed no influence of 25(OH)D on Δ BMD.	Adequate
Public	100% female			IG1: 48.3 (20.9)
	IG1: 59.9 (6.2) y			CG: 43 (16.6)
	CG: 61.2 (6.3) y			3 mo 800 IU D3 IG1: 70.8 (95% CI 66.4–76.1)
	100% African American			3 mo 2000 IU D3 IG1: 86.9 (95% CI 80.1–94.1)
				CG: no significant change
				RIA
Cooper (2003)120	187 Post menopausal women not on HRT		IG1: 10,000	25(OH)D	BMD: LS, FN, Ward's triangle, Tr, proximal forearm (DXA)	NR	4
Australia	IG1: 20/93 (21.5%), CG: 14/94 (14.9%) did not complete		IU Vit D2/wk + 1000 mg Ca/d	IG1: 82.6 (27.0)		No significant correlation between baseline 25(OH)D concentration and Δ BMD at any site or between Δ 25(OH)D and Δ BMD at any site.	Unclear
Public and Private	100% female		CG: 1000 mg Ca/d	CG: 81.6 (24.4)
	IG1: 56.5 (4.2) y		2 y	RIA
	CG: 56.1 (4.7) y
	Caucasian
Dawson-Hughes (1995)118	247 Healthy, ambulatory postmenopausal women		IG1: 700 IU	Baseline: NR	BMD LS, FN and total body (DXA)	NR	3
US	IG1: 5/128 (4%), CG: 8/124 (6%) did not complete		D3 + 500 mg Calcium citrate malate	End of study		25(OH)D concentrations during either season did not correlate with Δ BMD at any site.	Unclear
Public and private	100% female		CG: 100 IU	IG1: 100.1 (24.5)
	IG1: 63.0 y		D3 + 500 mg Ca daily	CG: 66.3 (25.5)
	CG: 64.0 y		2 y	Difference in means: 33.8 (95% 27.6, 40.1)
	Caucasion			CPBA
Ooms (1995)119	348 Elderly women		IG1: 400 IU	25(OH)D	BMD: FN, Tr and distal radius (DXA)	Season	4
The Netherlands	IG1: 51/177 (28.8%)		D3 CG: placebo daily	Median (25th and 75th percentiles):		Effect of vitamin D supplementation was independent of baseline 25(OH)D as well as 25(OH)D corrected for season.	Unclear
Public	CG: 53/171 (31.0%)		2 y	IG1: 27 (19–36) CG: 26.0 (19–37)
	100% female			1 y followup:
	IG1: 80.1 (5.6) y			IG: 62 (52–70)
	CG: 80.6 (5.5) y			CG: 23 (17–31)
	NR			CPBA
Schaafsma (2002)121	85 Healthy, postmenopausal women 50 – 70 y		IG1: eggshell powder + 200 IU D3 IG2: Ca carbonate + 200 IU D3 CG: placebo	25(OH)D	BDM: LS, hip (DXA)	NR	4
The Netherlands	12/85 (14%) did not complete		12 mo	IG1: 97.1 (24.1)		No significant correlation between 25(OH)D and BMD.	Unclear
NR	100% female			IG2: 83.1 (22.4) CG: 91 (36.5)
	IG1: 60.5 y			% change:
	IG2: 59.5 y			IG1: 25.1 (29.8)
	CG: 63.5 y			IG2: 43.8 (27.3)
	Caucasian			CG: 11.1 (22.7)
				CPBA
Storm (1998)116	60 Postmenopausal women without osteoporosis		IG1: 4 glasses of fortified milk (325 IU of vitamin D/quart)	25(OH)D	BMD: Tr, FN, LS (DXA)	Independent variables: Ca intake, 25(OH)D, bone markers, PTH, insulin growth factor I, age, BMI, thiazide use, smoking, and baseline BMD	4
The Netherlands	7/60 (12%)		IG2: Ca carbonate	Mean (SE): IG1: 63.5 (8)		Serum 25(OH)D was not a significant determinant of FN BMD at baseline, during winter (p=0.23) or over the entire study period.	Unclear
Public	100% female		CG: placebo daily	IG2: 68.8 (7.3)
	IG1: 71 y		2 y	CG: 59.8 (6.8); levels dropped almost 20% during 2 winters and returned to baseline during summer
	IG2: 72 y			End of study mean (SE): pooled: 67.8 (3.5)
	CG: 71 y			CPBA
	Caucasian
Prospective Cohorts
Bischoff-Ferrari (2005)132	327 Individuals with knee OA		1 – 2 y	25(OH)D	BMD FN (DXA Lunar DPX-L)	Adjusted for age, sex, BMI, knee pain, physical activity, cohort and disease severity.
U.S.	64% female			69.5 (30.5) nmol/L		Significant positive association between 25(OH)D and BMD independent of age, sex, BMI, knee pain, physical activity, and disease severity.
Public	228 complete data			% with values< 37.5 nmol/L: 15%		Significant trend between being in a higher serum 25(OH)D group and having higher BMD (p<0.04)
	74.4 (11.1) y			% with values 40–80 nmol/L: 51%
	Females: 76.6 (9.9) y			% with values > 80 nmol/L: 34%
	Men: 70.6 (12.1)			RIA
	NR
del Puente (2002)129	139 Active, non-institutionalized females (109 menopausal and 30 pre-menopausal)		2 y	25(OH)D	BMD LS and FN (DXA)	Adjusted for age, menopausal status, current smoking status and BMI.
Italy	124 at followup			Age 45–49 y: 57.7 (14.7)		25(OH)D independent predictor of BMD change at FN and LS (FN Δ BMD (beta 0.26 (0.13), p=0.04 and LS Δ BMD (beta 0.07 (0.03), p=0.04).
Public	15/139 (11%) did complete			Age 50–59 y -59.2 (19.2)		In stepwise analysis discrimination models only FN significant (partial R2=0.26, p=0.04).
	100% female			Age 60–69 y: 54.2 (16.7)
	58 (9) y			Age 70–79 y: 54.5 (19)
	Caucasian			<37.5 nmol/L: 17.3%; (range 9.1 to 27.5% across age groups).
				CPBA
Dennison (1999)127	316 Healthy adults age 60–75 y		4 y	NR	BMD: LS and proximal femur (DXA)	Adjusted for adiposity
U.K.	All 316 included in analysis			CPBA		No association between baseline 25(OH)D and BMD at LS and proximal hip (beta=0.002 spine, 0.001 hip) and no association between 25(OH)D and bone loss after adjustment for adiposity.
Public	45% female
	Women: 65.6 (2.8) y
	Men: 66.1 (3.2) y
	NR
Gerdhem (2005)131	1,044 Ambulatory independently living women		3 y	25(OH)D	BDM: FN and LS (DXA)	NR
Sweden	58/1044 (6%) did not complete			95 (30)		No association between baseline 25(OH)D and BMD.
Public	100% female			% with values < 50 nmol/L: 4.4%		See other tables for other outcomes
	75 (75–75.9) y			% with values < 75 nmol/L: 26%
	NR			CPBA
Melin (2001)126	64 Healthy, independent elderly individuals		1 y	25(OH)D	BDM: FN (DXA)	Adjusted for BMI
Sweden	All 64 included in analysis			Outdoor exposure ≥ 3 h/wk (N=49); males: 67.5 (15) females: 60 (27.5) nmol/L.		FN BMD associated with serum 25(OH)D after summer (r=0.38, p=0.003) and winter (r=0.37, p=0.003). After adjusting for BMI, 25(OH)D remained a significant determinant after winter (adjusted R2=0.14, p=0.005).
Public	81% female			Indoor exposure < 3 h/wk females (N=14): 40 (12.5)
	83.7 y			% with values < 77.5 nmol/L: 78%
	Caucasian			RIA
Rosen (1994)135	18 Healthy independently living elderly women		2 y	25(OH)D	BMD LS and FN (DXA)	NR
U.S.	3/18 (17%)			Baseline: 72.5 (6.7)		Δ 25(OH)D between summer and winter was associated with LS BMD in 2nd y (r=0.59, p=0.04) but not FN BMD.
Public	100% female			6 mo: 63 (3)
	77 (2) Y			12 mo: 88 (7.8)
	NR			18 mo: 70.9 (8.5)
				CPBA
Stone (1998)128	261 Health elderly females > 65 y		42 – 71 mo	25(OH)D	BMD TH (DXA) calcaneal (SPA)	Adjusted for age, weight, clinic site, current use of Ca supplements, multivitamins containing vitamin D, physical activity, smoking status and season. Controlled for levels of other hormones.
U.S.	random sample -subcohort of individuals not on HRT from Study of Osteoporotic Fractures			65.5 (24.5)		Significant association between lower 25(OH)D levels and TH BMD loss. Lower 25(OH)D levels associated with increased loss at TH after adjusting for estradiol, testosterone, and SHBG, season, and use of supplements.
Public	30/261 (11%) without calcaneal BMD; 43/261 (16%) without hip BMD			RIA		25(OH)D not associated with calcaneal BMD after adjusting for age and weight.
	100% female
	71.3 (4.8) y
	Caucasian
Case-control studies
Al-Oanzi (2006)152	56 Men with idiopathic osteoporosis	NR		25(OH)D3	BMD diagnosis of osteoporosis based on T-score FN and LS	NR
U.K.	114 male ctrls			Cases: 44.7 (21)		No significant difference between plasma 25(OH)D in cases and ctrls, but mean free plasma 25(OH)D was about 33% lower in men with OP vs. ctrls (p<0.0001).
Public	100% male			Ctrls: 43.3 (17)
	Cases: 59.6 (13.6) y			RIA
	Ctrls: 62.4 (10.4) y
	Caucasion
Boonen (1999)139	100 Postmenopausal women	Age, PM status, sampled at same time of year		25(OH)D	BMD FN and Tr (DXA) Fractures	Adjusted for age
Belgium	50 hip fracture patients, 50 ctrls			Cases 29.25 (26.5)		Mean 25(OH)D3 was lower in cases vs. ctrls (p<0.001).
Public	100% female			Ctrls: 68.75 (39)		Vitamin D deficiency (< 30 nmol/L): 64% of cases vs. 8% ctrls within the same 4 mo sampling period (no relation b/w 25(OH)D and mo of sample collection). FN and Tr BMD were significantly lower in cases than ctrls. No significant relation found b/w the 25(OH)D3-PTH axis and BMD in cases and ctrls. In multiple regression of pooled data, models using 25(OH)D3 and PTH were highly predictive of FN BMD (R2=32%, p < 0.001).
	Cases: 74.2 (7.8) y			% with values < 30 nmol/L
	Ctrls: 75.8 (5.6) y			cases: 64%
	NR			ctrls: 8%
				CPBA
Landin-Wilhelmsen (1999)140	128 PM osteoporotic pts, 227 age matched ctrls from outpatient clinic	Age, gender, PM status		25(OH)D3:	BMD and BMC: LS,	NR
Sweden	100% female			Cases: 88 (30)	TB and FN (DXA)	25(OH)D significantly lower in OP pts vs. ctrls (p<0.05).
Public	Cases 59 (6) y			Ctrls: 96 (32)	Fractures	OP pts had lower body weight and BMI vs. ctrls (p<0.001).
	Ctrls 59 (5) y			RIA
	NR
Villareal (1991)143	98 Ambulatory, independently living PM women 49 women with low (<38 nmol/L) 25(OH)D and 49 Ctrls.	Age, gender, PM status, ethnicity, season, independence status, geographical location		Cases: 23 (7)	BMD (LS, T12-L3) QCT	NR
U.S. (Mid West)	100% female			Ctrls: 58.9 (19)	iPTH (RIA)	Women with low 25(OH)D levels had a reduced LS BMD. In the low 25(OH)D group, LS BMD correlated with 25(OH)D (r=0.41, p < 0.01).
NR	Cases: 64 y			CPBA		In multivariate analysis, iPTH was the major determinant of a decrease in LS BMD.
	Ctrls: 63 y
	Caucasion
Thiebaud (1997)141	179 Hip fracture patients (136 women and 43 men) 180 hospital ctrls (136 women and 44 men) 55 community ctrls (47 women and 8 men)	Age, setting (for cases and one control group)		25(OH)D	BMD FN, TH and Tr (DXA)	Adjusted for age, sex, and creatinine 25(OH)D levels generally low especially in hospital ctrls and hip fracture cases.
Switzerland	% female hip fracture cases: 76%			Fracture cases:	Fractures	Women and men with hip fractures significantly lower 25(OH)D levels vs. ctrls. Fracture patients had lower hip BMD vs.ctrls (p < 0.001).
Public	hospital ctrls: 76%			women 25.5 (20.5)		Significant biochemical markers in the multivariate logistic regression model of the risk for hip fracture were serum albumin and PTH.
	community ctrls: 85%			men 17.25(18.5)		In women FN, Tr BMD weakly correlated with 25(OH)D and the only significant association was at the Tr (r=0.13, p < 0.05).
	Cases: 81.0 y (women) and 77.7 y (men); Hospital ctrls: 80.9 y (women) and 76.9 y (men); Community ctrls: 71.7 y (women) and 71.3 y (men)			Hospital ctrls:
	NR			women 31.5 (26.5)
				men 27.75 (21.5) Community ctrls:
				women 53(23)
				men 31.5 (22.8)
				RIA
Yan (2003)136	352 Older individuals (60–83 y)	Age, ethnicity		Chinese men 27.1 (11.5), women 30.9 (13.5); and British men: 36.6 (12.1), women 34.7 (13.7)	BMC: FN (DXA)	Adjusted for bone area, weight, height, age and sex
China 42° N and U.K. 52 °N	% female			% with values <25 nmol/L:		Significantly higher 25(OH)D levels in British subjects. Weak association (r=0.054, p=0.05) b/w 25(OH)D and FN BMC in British subjects after adjusting for size but not in Chinese subjects.
Public	Chinese: 50.5%			Chinese: men 53%, women 39%; British: men 20.9%; women 28.4%.
	British: 50%			RIA
	Chinese:
	male 67.9 (3.6) y
	female 65.2 (3.7) y
	British:
	male 69.1 (6.1) y
	female 68.2 (6.5) y
	64% Chinese (Asian), 36% British (Caucasion)

total 25(OH)D or either isoform of 25(OH)D (isoform not specified);

Δ

, change in; b/w, between; ctrls, controls; AC, allocation concealment; DXA, dual-energy X-ray absorptiometry; FN, femoral neck; IA, immunoassay; NR, not reported; OA, osteoarthritis; OP, osteoporosis; N, north; PTH, parathyroid hormone; QCT, quantitative computed tomorgraphy; RIA, radioimmunoassay; S, south; TH, total hip; Tr, trochanter; vit, vitamin; y, year;