Consensus statement from 2^nd International Conference on Controversies in Vitamin D

Vitamin D deficiency and associated health risks are problems that still need to be managed and addressed worldwide. Generally, dietary intake recommendations are not met. The calculation of dietary intake is based on a combination of content of vitamin D in our food and information on the amount of food consumed.

Analytical data for vitamin D in food samples are no better than the sampling strategy and the performance of the analytical methods. Due to the development of simpler and cheaper chemical analyses of vitamin D, information on vitamin D content in food has markedly increased in the the last 5-10 years. In food, vitamin D activity derives from the parent forms vitamin D3 and vitamin D2, and the corresponding hydroxylated forms 25(OH)D3 and 25(OH)D2.

The best available vitamin D sources are cod liver oil (90-250 μg/100g) and fatty fish, e.g. aquaculture salmon (6-10 μg/100g) and wild mackerel (5-8 μg/100g) [58‐61]. But as dietary intake depends on the amount consumed, food products with lower content also play a role. Thus, vitamin D3 and 25(OH)D3 are found in fish, eggs, meat, and dairy products [58]; vitamin D2 is found in wild mushrooms or cultivated mushroom exposed to ultraviolet B (UVB) rays, whereas beef and dairy products contain vitamin D2 and 25(OH)D2 [62‐64].

To calculate total vitamin D activity in food, conversion factors between the different vitamin D forms are essential [60]. The contribution to vitamin D activity from 25(OH)D3 compared to 25(OH)D2 has been assessed in human intervention studies [61‐63]. Studies that have compared the effect of dietary intake of vitamin D3 and vitamin D2 on vitamin D status have been evaluated in a systematic review and meta-analysis [64]. The overall conclusion is that when vitamin D is administered once or as a monthly bolus, vitamin D3 is superior to vitamin D2 in increasing vitamin D levels, whereas no difference in vitamin D status is observed if vitamin D2 and vitamin D3 are administered on a daily basis [64]. The only human intervention study performing a randomized cross-over design examining the difference in vitamin D status by administration of daily vitamin D3 and vitamin D2 found vitamin D3 to be superior in maintaining vitamin D status [63].

Foods can be fortified with vitamin D and, in this setting, would be referred to as bio-fortified. In the European Union, the beneficial effects of this strategy are limited due to a restriction of the maximum content of vitamin D in feed for livestock [65]. However, apart from this limitation, it is still possible to increase the content of vitamin D in food of animal origin. In Denmark, for example, the current recommendation is 400 IU/kg feed to slaughter pigs. If this limit was increased to the maximum EU level of 2,000 IU/kg feed, the content of vitamin D in the food product would increase by a factor of 3 [66]. In eggs, vitamin D may be increased 10-fold compared with the current maximum level, without harming hens. In this example, one egg could contain the entire day’s nutritional recommendation [67].

Intervention studies have shown the possibility of increasing dietary intake with dietary supplements, enriched food and bio-fortified food. Some countries have chosen to introduce mandatory fortification (e.g. Finland), while other countries’ foods are enriched, but not through legislation (e.g. USA). Other countries continue to be engaged in a debate as to what strategy is best for their needs.

Solar UVB (~295-315 nm) light is the main source of vitamin D for most human beings. It is well known that at a given latitude individuals with darker skin have poorer vitamin status [25(OH)D3] than those with lighter skin. Latitude is a determining factor for the extent of UVB exposure. Differences in exposure to this wavelength of light are usually attributed to the photoprotective (i.e. inhibitory) properties of melanin. However, studies investigating the role of melanin are inconsistent. The impact of melanin on vitamin D status was compared across a range of skin types (white to black) after whole body exposure with two different solar-range Ultraviolet Radiation (UVR) spectra. UVR exposures were fixed across skin types for a given UVR source and were fixed to be sub-erythemal; thus, the only clear variable was skin color. Repeated exposures were given at 3-4 day intervals and blood samples were taken before irradiation and after the final irradiation for the assessment of 25(OH)D3. The UVR dose response curves for 25(OH)D3 were linear and the ratio of the slopes of black and white skins were compared to estimate a melanin protection factor, which was <1.5 with both UVR sources. This result shows modest inhibition of vitamin D production by melanin, but it is insufficient to explain the epidemiological differences in different skin types.

Sunscreens are effective because they attenuate solar UVR. Their use can inhibit cyclobutane pyrimidine dimers (CPDs) [68], sunburn and skin cancer [69]. The sun protection factor (SPF) of a sunscreen is primarily an index of its ability to attenuate UVB [70]. Thus, in theory, sunscreen use would be expected to inhibit the production of vitamin D. However, there are conflicting views about whether this is the case in practice because sunscreens are typically used sub-optimally [71, 72]. This could be due, at least in part, to the need to reapply such screens at regular intervals to maintain the protective effect or incomplete skin covering with sunscreen.

By definition, the higher the UVA protection factor (UVA-PF) for a given SPF, the lower the UVB protection. Two SPF = 15 sunscreens used correctly during a week of perfect weather with a maximum daily UV index (UVI) of 9 (deemed very high) were compared with each other. In study by Petersen and colleagues [73], participants included one group with a low UVA-PF (n=20) and the other with a high UVA-PF (n=20), and instructed on how to use sunscreens to achieve the labelled SPF (i.e. apply 2mg/cm² skin). Another group (n=22) was told to bring their own sunscreens and use as they do typically, which might be expected to be ~0.8mg/cm² [73]. Participants in the typical use group had sunburn of 5 in exposed body sites. Sunburn was not observed in the sunscreen intervention groups. All groups had a highly significant increase in serum 25(OH)D3 at the end of the test period. The increase in the typical use group was greater than both sunscreen intervention groups, and the increase in the high UVA-PF group was greater than in the low UVA-PF group. These data show that even when sunscreens are used optimally to prevent sunburn under very high UVI conditions, they permit skin synthesis of vitamin D.

Consensus Statements:

Research agenda

Patients exposed to glucocorticoid excess have a two-fold higher risk of vitamin D deficiency compared to the general population, that is most likely related to the underlying disease and the direct effects of glucocorticoids on vitamin D metabolism [74].

It is still unclear whether these effects on vitamin D metabolism may occur clinically, since serum calcitriol [1,25(OH)D] values are variable in patients with glucocorticoid-induced osteoporosis (GIO) [75]. Moreover, vitamin D receptor expression may be decreased by glucocorticoid excess in several tissues and cells, leading to a vitamin D resistant state [76, 77]. These actions of glucocorticoids on vitamin D metabolism and activity may be negatively synergized by the underlying disease for which glucocorticoid treatment is given [78]. Based on these pathophysiological concepts, the use of calcitriol or alfacalcidol has been proposed instead of cholecalciferol for the treatment of hypovitaminosis D in glucocorticoid-induced osteoporosis [79].

Randomized studies have demonstrated that combination therapy with calcium and vitamin D was shown to be more effective in preserving bone mineral density than either calcium or no treatment with a weighted mean difference of 2.6% and 2.5% at lumbar spine and forearm, respectively [80]. Moreover, the administration of both vitamin D and calcium is expected to potentiate the anti-fracture effects of bone-active drugs in glucocorticoid-induced osteoporosis, such as already demonstrated in post-menopausal osteoporosis [81].

Therefore, vitamin D and calcium are recommended in patients exposed to therapeutic amounts of glucocorticoids.

In patients with CKD, alteration in vitamin D metabolism plays a central role in the development of secondary hyperparathyroidism (SHPT), in addition to being associated with increased cardiovascular morbidity and mortality [82]. A hallmark of vitamin D insufficiency/deficiency is elevated levels of parathyroid hormone and consequently most guidelines on the use of vitamin D in CKD have largely been based on levels of PTH and calcium. UVB exposure is effective in increasing the serum levels of 25(OH)D even in patients with end-stage kidney disease on dialysis [83].

Since the late 7O's, native vitamin D and nonselective vitamin D receptor (VDR) activators have been used mainly for lowering of PTH levels [84, 85]. In the past two decades, selective VDR activators have gained recognition for their importance in the management of CKD-mineral and bone disorder (MBD) and as such are considered standard therapy in these patients. More recently, vitamin D deficiency has been linked to a whole host of diseases, some related to CKD, prompting further exploration of the mechanism of action of active vitamin D analogues and, consequently, their potential benefit in clinical trials. However, many open questions regarding the use of native vitamin D or VDR activators remain [86‐89]. First, the Kidney Disease Outcomes Quality Initiative (K/DOQI) and Kidney Disease: Improving Global Outcomes (KDIGO) guidelines recommend testing for vitamin D insufficiency and deficiency in CKD patients, but no consensus on the definition of vitamin D insufficiency in CKD is currently available [90]. Second, using native vitamin D in patients with CKD remains heavily debated, particularly with regard to the choice of compound, when to treat, and the most suitable dose to administer with or without VDR activators [89].

Interestingly, 25(OH)D serum levels <37.4 nmol/L have been repeatedly reported in up to 50% of CKD 5 stage patients [91], while severe vitamin D deficiency seems to be present in approximately 95% of patients on hemodialysis [92]. The same findings have been reported for kidney transplant recipients, even in those with relatively well-preserved renal function [93]. Together with these findings, vitamin D deficiency has been associated with several complications of CKD, such as cardiovascular disease, anemia, proteinuria, progression of renal failure and disordered calcium metabolism [89, 94]. After kidney transplantation, low levels of 25(OH)D serum levels are the most important predictor of the sustained increase in PTH levels, which persists in up to 50% of patients even many years after surgery [93].

In a six-month prospective controlled study, Molina et al. [95] showed that cholecalciferol was able to reduce albuminuria even after controlling for a range of potential confounders. In a recent randomized, double-blind, placebo-controlled trial, cholecalciferol supplementation was associated with an improvement in vascular function in non-diabetic CKD 3-4 stage patients [96]. After renal transplantation, cholecalciferol administration has been found to significantly reduce PTH levels. Finally, a recent randomized, double-blind, placebo-controlled study from Yadav et al. [97], in which cholecalciferol was given to patients with CKD stages 3-4 before transplantation, PTH levels declined significantly. The authors also showed a substantial decrease in bone alkaline phosphatase activity and C-terminal cross-linked collagen type I telopeptide, strongly suggesting that the cholecalciferol­ induced decrease in PTH may reduce bone remodelling in these patients.

Consensus Statements:

One of the more intriguing aspects of the pleiotropic effects of vitamin D is its putative relationship to cancer [108, 109].

Preclinical cell and animal studies provide a compelling story and a strong rationale for a benefit of vitamin D and avoidance of vitamin D deficiency for cancer risk and outcome [108, 109]. Human observational and association studies are mixed regarding the benefits of vitamin D, but avoidance of vitamin D deficiency seems to more clearly indicate a health benefit [108, 109]. Several RCTs were discussed at the meeting especially the ViDA trial [110], but the results of other RCTs were not all available until they were published after the meeting. The trials generally concluded that risk of developing cancer was not statistically reduced by vitamin D supplements [110‐112]. However, potential weaknesses in these studies included the fact that the vitamin D dose may not have been high enough and especially the time of follow-up was not long enough to see a reduction in the risk of developing cancer. It is important to emphasize that the control group receiving a placebo was not vitamin D deficient; thus studies tended to compare subjects with adequate circulating vitamin D concentration to subjects treated with supplements that achieved higher levels of circulating 25(OH)D.

In the recently published meta-analysis of the effect of vitamin D on incidence and survival of cancer patients, data also showed no significant reduction in cancer incidence [112]. However, vitamin D supplementation in highly significant findings reduced total cancer mortality. For total cancer mortality, five trials were included in the analysis with 1591 deaths; 3-10 years of follow-up; and 54-135 nmol/L of attained levels of circulating 25(OH)D in the intervention group. The summary RR was 0.87 (95% CI, 0.79-0.96; P = 0.005; I2 = 0%), which was largely attributable to interventions with daily dosing (as opposed to infrequent bolus dosing).

In a recent nested case-cohort study from Japan, a lower overall risk of cancer was found in men and women with higher levels of 25(OH)D [113]. Many previous studies have investigated the association of vitamin D with specific tumors. For example, cancers of the liver have been found to be associated with low 25(OH)D levels in cohort studies. In some studies, no association of vitamin D was found with prostate cancer, but there was a strong association between low vitamin D levels and high-grade prostate cancers [114].

CYP27B1, the final activating enzyme in the synthesis of calcitriol, is not only present in the kidney, but also expressed in non-renal sites as is the VDR. These non-renal sites include tumor cells themselves as well as cells in the tumor microenvironment [115].

Inhibition or mutations that inactivate the CYP24A1 enzyme lead to excessive 1,25(OH)2D production and hypercalcemia. Use of CYP24 inhibitors such as ketoconazole, liarazole and genistein among others have been shown to increase the anti-cancer activity of vitamin D in cell and animal models [116]. A recent study used a designed small molecule inhibitor of CYP24A1 known as CTA091 [117]. To avoid the systemic effects, a tumor targeted nanoparticle delivery system was developed to treat epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) lung cancer resistant to erlotinib. Delivery of vitamin D based drug payloads with CYP24A1 inhibitors via a tumor-targeted system was effective without causing side-effects and appears to be promising as a new therapy for EGFR TKI resistant lung cancer.

The goals of endocrine therapy of estrogen receptor positive (ER+) breast cancer are to deprive the cancer of the driving force for growth, namely estrogens. Three clinical approaches include: 1) using aromatase inhibitors (AIs) to prevent estrogen synthesis; 2) using selective estrogen receptor modulators (SERMs) to block estrogen binding to the ER; and using down-regulators of the ER (SERDs) to decrease the tumor concentration of ERs. Calcitriol has been shown to mediate all 3 activities [118]. Calcitriol or dietary vitamin D rapidly and equivalently inhibited the growth of mouse and human breast cancer in various mouse models [119, 120].

Vitamin D or its analogues demonstrate cell autonomous activity directly by acting via the VDR in tumor cells. Vitamin D can also regulate tumor cell behavior indirectly by acting on various cells in the microenvironment including stromal cells, adipose tissue and inflammatory cells, etc. Actions on the breast micro­environment contribute to vitamin D effects to inhibit breast cancer cell growth [121, 122]. In studies of colon cancer [123] and pancreatic cancer [124], an important additional anti-cancer activity of vitamin D via unique effects on cells in the micro-environment was demonstrated.

In experiments with the non-metastatic breast cancer cell line FARN168, studies showed that knocking down the VDR allowed the cell to transition into a metastatic cell [125]. Further evaluation demonstrated that restoring the VDR to the metastatic cells (VDR rescue) returned the cells to the non-metastatic state. Overexpression of the ID-1 gene, a gene commonly overexpressed in cancer cells, was identified as overexpressed in the VDR knockdown cells and reduced in the tumor cells when VDR expression was rescued suggesting that regulation of ID-1 by vitamin D contributes to its action to improve survival by inhibiting metastasis.

In a small trial including 29 cancer patients in which subjects received either high or low dose (placebo) vitamin D supplements, vitamin D supplementation was able to decrease circulating levels of 27-hydroxycholesterol, a recently identified endogenous SERM that can stimulate proliferation of breast cancer cells [126]. This study hypothesized yet another anticancer mechanism of vitamin D, acting by inhibition of the synthesis of a breast stimulatory endogenous SERM, likely by CYP27A1 inhibition.

A role of vitamin D has also been observed in colorectal cancer [127]. To identify optimal concentrations for colorectal cancer risk reduction, the association between circulating 25(OH)D and subsequent colorectal cancer incidence was studied in 17 prospective cohorts participating in the international Circulating Biomarkers and Breast and Colorectal Cancer Consortium [127]. Pooled data were correlated with 25(OH)D measurements obtained prior to cancer diagnosis. These findings demonstrated a strong, statistically significant, and robust inverse association between pre-diagnosis circulating vitamin D and colorectal cancer risk that was taken to substantially strengthen the evidence, previously considered inconclusive, for a causal relationship between circulating 25(OH)D and colon cancer risk. The study suggests that optimal circulating 25(OH)D concentrations for colorectal cancer risk reduction are 75-100 nmol/L, values somewhat higher than current Institute of Medicine in USA (IOM) recommendations for bone health.

Consensus Statements:

Research agenda

Celiac disease (CD) is an autoimmune disorder of the gastrointestinal tract due to an immune response against gluten-containing grains in genetically susceptible individuals [129] with genotypes encoding the Human Leukocyte Antigen (HLA) class II molecules HLA-DQ2 and HLA-DQ8 [130]. The two markers of CD are a reduction in villous surface area of the small intestine and the appearance of anti-transglutaminase-2 antibodies in the plasma. Intercellular tight junctions are also altered in CD [131‐133].

Low vitamin D levels have been linked to aberrant immune function in gastrointestinal diseases, including CD, inflammatory bowel disease (IBD) and dysbiosis [134, 135]. The vitamin D receptor (VDR) is highly expressed in the intestine. The activated VDR regulates innate immune response, promotes immune tolerance in the gut and also plays a role in maintaining intestinal barrier function, through regulation of the expression of the tight junction proteins. Vitamin D also regulates the gut microbiota, by controlling the composition of the gastrointestinal microflora [136, 137]. Specifically, data from animal models suggest that in the absence of the VDR or the ability to produce 1,25(OH)2D, unregulated inflammation of the gut results in an environment that supports the expansion of noxious bacteria in the Proteobacteria phylum, including the Helicobacteraceae family members, that out-compete beneficial members of the Firmicutes and Deferribacteres phyla.

Consensus Statements:

Research agenda

The prevalence of diabetes mellitus among patients treated for osteoporosis varies widely, from less than 10 % in Europe to more than 25 % in some US populations [138].

Increased fracture risk was observed in both type 1 (T1DM) or type 2 (T2DM) diabetes. The rate of hip fracture risk is up to 7-fold higher in patients with T1DM and about 1.3-fold higher in patients with T2DM. It is probably due to detrimental effects of impaired glucose metabolism on bone health as well as to an increased risk of falls or other traumatic events, frequently reported in diabetic patients [139, 140].

Changes in mineral bone density (BMD) are not similar in T1DM and T2DM patients, and often conflicting. In the Melbourne Collaborative Cohort Study, vitamin D status was inversely associated with the risk of T2DM, and this association did not appear to be explained by reverse causality [141].

A potential role for abnormal vitamin D status in changes of glucose homeostasis has been described [142]. It has been demonstrated that vitamin D deficiency is detrimental to the synthesis and secretion of insulin in animal and human studies. In several, but not all, human observational trials, an inverse correlation was seen between vitamin D with insulin insensitivity, prediabetic states and dysglycemia. Recently, data from non-interventional observational studies have shown a negative relationship between the vitamin D status and parameters of insulin insensitivity and incidence of T2DM [143]. In a meta-analysis of 21 studies, the association between vitamin D and parameters of insulin insensitivity and incidence of T2DM were demonstrated [144].

From a pathophysiological point of view, vitamin D levels and thioredoxin interacting protein were associated with different beta-cell dysfunction markers, indicating their potential abilities to predict the beta-cell status in people with diabetes [145].

Evidence from observational studies as well as recent clinical trials support the beneficial effect of vitamin D on glycemic control and subsiding systemic/vascular inflammation in T2DM [146, 147]. Moreover it was showed that diabetic subjects might respond differently to vitamin D supplementation according to their VDR Fokl genotypes [148].

Vitamin D supplementation is beneficial for the reduction of hs-CRP in T2DM subjects but does not have a significant influence on Tumor Necrosis Factor α (TNF-α) and interleukin – 6 (IL-6) in T2DM subjects [149].

With regard to the effect of vitamin D supplementation on fasting plasma glucose (FPG), insulin resistance and prevention of T2DM in non-diabetics, no significant effect was observed on controlling FPG level, improving insulin resistance or preventing T2DM in non-diabetic subjects in a pooled meta-analysis [150]. On the other hand, vitamin D administration and improved vitamin D status was shown to improve glycemic measures and insulin sensitivity.

Vitamin D deficiency is highly prevalent, especially in elderly obese individuals [48], with proven detrimental effects on bone and muscle health. Considering the inverse correlation between BMI and vitamin D levels and the large prevalence of obesity worldwide, vitamin D deficiency is a growing health concern in the obese, regardless of age group [151]. Adipose tissue is a direct target of vitamin D, which plays a role in modulating adipose tissue distribution and activity [152]. This is further confirmed by evidence of vitamin D receptors (VDR) in pre -adipocytes, adipocytes, in both subcutaneous and visceral adipose tissue [153]. Despite several epidemiological studies showing the existence of a close relationship between obesity and hypovitaminosis D, the mechanisms underlying this association are still largely unknown. Interestingly, it has been suggested that vitamin D may provide a protective effect in obese individuals, by reducing systemic inflammation. Therefore, vitamin D may be considered a protector for obesity and related clinical conditions. However, only a few and mostly underpowered randomized clinical trials have been conducted to test the effectiveness of vitamin D supplementation in facilitating weight loss or other metabolic outcomes in obese people. Similarly, recent evidence linking vitamin D deficiency to non-alcoholic fatty liver diease (NAFLD) progression has led to the hypothesis that vitamin D may play a protective effect, controlling hepatic inflammation, with decreased liver mRNA expression of resistin, IL-6 and TNF-a [154]. However, also in this case, clinical trials have yielded inconsistent results.

A recent meta-analysis of 35 studies (including 17,245 persons) showed that serum vitamin D is inversely associated with body fat mass (FM), but the analysis of RCTs does not support the hypothesis that vitamin D supplementation augments body-fat loss [155].

Bariatric surgery has been proven to be the most effective treatment of morbid obesity, leading not only to a long-term weight reduction but also to a significant improvement of health-related quality of life and a reduction of overall mortality.

Biliopancreatic diversion (BPD) procedure causes the most severe and persistent reduction in serum 25(OH)D. A retrospective study revealed that 25(OH)D levels decrease over time with BPD even 9 years after surgery [156]. Therefore, prevention and treatment of hypovitaminosis D in patients undergoing bariatric surgery is crucial in order to prevent bone loss and other complications. Regardless of bariatric surgery procedure choice, all patients need periodic assessment of their vitamin D status.

Consensus Statements:

Research agenda

Multiple sclerosis (MS) is an autoimmune disease that alters the central nervous system, leading to the presence of focal areas of inflammation and demyelination [157]. Increasing evidence suggests that several environmental factors including inadequate vitamin D levels are associated with the progression of MS [158]. Individuals with adequate levels of vitamin D appear to have reduced prevalence, activity and progression of MS [159]. Several observational studies have explored factors affecting vitamin D levels (e.g. sunlight exposure, latitude and diet) and support an association between elevated vitamin D levels and reduced MS disease severity [160, 161]. In experimental studies examining the effect of vitamin D supplementation, it has been observed that low serum vitamin D levels can exacerbate MS symptoms and are associated with higher relapse rates, new lesions and greater disability [162‐165]. Furthermore, daily cholecalciferol supplementation has recently been shown to improve depressive symptoms in patients with relapsing remitting MS [166]. Lower vitamin D levels were associated with higher depressive scores and vitamin D supplementation improved these symptoms [166].

The majority of studies in the MS setting suffer from substantial heterogeneity in terms of study design, baseline serum vitamin D levels and outcome measures, making cross-comparison difficult and results inconclusive. To address these shortcomings, systematic reviews have been undertaken to examine the effect of vitamin D supplementation on MS; however, some of these have been limited by lack of a range of vitamin D status and not accounting for bias [167] or failure to assess cytokine outcomes or baseline vitamin D on outcomes [168]. A third systematic review has attempted to address some of these former weaknesses [169]. In this meta-analysis which included 10 studies, vitamin D supplementation was compared to placebo or low dose vitamin D. Overall, disease measures improved to a greater extent in patients with lower baseline serum 25(OH)D levels. In 3 out of the 10 studies included, an improvement in disease measures was more apparent in patients with lower baseline vitamin D levels.

A number of Mendelian randomization studies identified a link between genetically low vitamin D status and MS (n=95) and another autoimmune disease, T1DM (n=1). This makes a causal link between vitamin D status and these diseases likely, particularly for MS.

Mounting evidence places vitamin D as a central and necessary nutrient for conception, normal placental function, and maternal and fetal immune homeostasis. It is also a key factor for continued well-being during lactation [170, 171]. Outcome data from 4 randomized controlled trials during pregnancy and lactation [172, 173] as well as other studies [174, 175], collectively support the Barker hypothesis [176, 177] that identifies vitamin D as a vital factor in maternal and infant well-being.

Alluding to general findings that skin color is important in vitamin D homeostasis, women most affected by suboptimal vitamin D status are those of darker skin pigmentation or with limited sunlight exposure. In the US, for example, African American women have the most significant vitamin D deficiency and the worse pregnancy outcomes. While many other factors could account for worse pregnancy outcomes among African American women, the potential negative impact of vitamin D deficiency looms large as a contributing factor [178]. After pregnancy, the need for adequate vitamin D in mother's milk ensures not only adequate vitamin D delivery to the breastfeeding infant, but it also ensures that immune competence aided by vitamin D is optimized through mother's milk [170, 179]. Ensuring adequate vitamin D nutrition during pregnancy and lactation should be an integral component of global public health policies.

Consensus Statements:

Define the optimal vitamin D status before conception, during pregnancy and lactation for optimal health of the mother and her offspring.

Accumulating evidence indicates that vitamin D hormone may play a role in aging and age-related cognitive decline [180, 181]. Human studies have noted a positive association between vitamin D levels and cognitive functions in aged individuals. Annweiler and colleagues reported that supplementing elderly individuals with vitamin D during 16 months improves executive function [182, 183]. Several neurological disorders have been possibly connected with deficient vitamin D status, including multiple sclerosis [184], Parkinson's disease [185], and Alzheimer’s disease (AD) [186].

In animals, vitamin D deficiency during the early stages of life has a strong influence on brain function when maturity is reached, suggesting that vitamin D deficiency, at different time-points and for varying periods of time, could be a component of neurodegenerative diseases such as Alzheimer's disease [180, 187]. In addition, in vitro and in vivo experiments have demonstrated the following about vitamin D neurophysiology: 1) upregulation of the expression of several neurotrophins; 2) stimulation of the secretion of anti- inflammatory cytokines; 3) modulation of neurotransmitters such as acetylcholine, dopamine and serotonin; 4) regulation of growth factors such as glial cell-derived neurotrophic factor and neurotrophin nerve growth factor. It has also been observed that vitamin D is involved in 1) synaptogenesis, 2) axonal growth, 3) intraneuronal calcium homeostasis, and 4) inhibition of nitric oxide synthase [188].

Animal models of AD and in vitro studies have demonstrated that vitamin D 1) enhances cerebral clearance of human amyloid peptide from mouse brain across the blood -brain barrier and 2) prevents amyloid- induced alterations in cortical neurons [189]. Vitamin D3 deficiency increases spatial learning deficits in these animal models. Conversely, vitamin D3 supplementation decreases pathological markers of the disease, such as amyloid deposition, but also alters the expression of various genes, some of which are involved in the regulation of estrogen and insulin-like growth factor 1 (IGF-1) production [190, 191].

Hippocampus-dependent learning and memory formation are associated with increased cell proliferation and neurogenesis. A reduction of hippocampal neurogenesis has been shown to be involved in the pathogenesis of AD [192]. In this regard, vitamin D appears to improve working memory and endogenous neurogenesis, but only when presented before the onset of the major symptoms. Vitamin D deficiency, on the other hand, increases the amyloid burden and impairs neurogenesis in the brains of male transgenic animals [193]. Thus, these studies suggest critical windows of time, hormonal interactions, and gender-specific effects of the processes in which vitamin D may be important [190, 191]. This conclusion is supported by additional studies between male and female animals with AD [190, 191, 193]. In these studies, vitamin D supplementation was observed to induce a better cognitive performance in late stages of female animals and in the early stages of males, but not in the late stages of male animals [190, 191, 193]. The exact molecular mechanisms by which vitamin D could be beneficial in AD are not yet well understood but collectively, the results suggest some role [193, 194].

Consensus Statement:

Pre-clinical and clinical studies are warranted to understand the role of vitamin D in neurological diseases.

Falls are complex events that often involve multiple deficits. Of the 10 leading causes of falls, the strongest predictors, in descending order are: 1) muscle weakness, 2) history of falls, 3) gait deficit, and 4) balance deficit [195]. Vitamin D deficiency mimics several of the age-related changes that occur in muscle and that are associated with increased risk of falling. In vitamin D deficiency, there is a preferential loss of type 2 muscle fibers [196], fewer intramuscular vitamin D receptors [196], and reduced balance [197]. Vitamin D has been shown to be associated with the prevention of a series of complications of chronic diseases, such as fracture and fall risks [198]. A recent meta-analysis of 29 randomized placebo-controlled intervention trials revealed that vitamin D supplementation improved muscle strength [199]. Vitamin D supplementation did not increase muscle mass or power but this evidence was limited to the few available studies that included these measurements [199]. Vitamin D supplementation has also improved balance in older adults, as measured by reduced sway [197].

The evidence so far evaluating the effect of vitamin D supplementation on reducing the risk of falls is mixed. A 2011 meta-analysis examining the role of vitamin D dose concluded that a dose of 700 to 800 IU was required to lower risk of falls and that with optimal dosing, the risk reduction was on the order of 15% [200]. Several trials then tested 800 IU per day. In one conducted in postmenopausal women, neither 800 IU per day (nor 50,000 IU twice monthly, also tested) influenced the risk of falling [201]. A similar negative result was observed in another 800 IU trial in over 400 women aged 70 to 80 years [202]. However, an intervention study testing multiple vitamin D supplement doses ranging from 400 to 4,800 IU per day found significantly fewer falls among postmenopausal women taking doses of 1,600 - 3,200 IU per day [203]. A limitation of this study was its relatively small sample size.

The ViDA trial that tested the effect 100,000 IU per month on fall risk in 5,108 adults over a 3.3-year period did not observe a significant effect. Outcomes of trials testing even higher, less frequent doses of vitamin D will be discussed in a different section. A factor that may affect efficacy of supplementation is the vitamin D status of the study population. The trials cited above involved participants who were vitamin D replete or only mildly insufficient [201, 202]. An exception to this was a trial conducted in Brazilian women who had a low mean serum 25(OH)D level of 37.4 nmol/L [204]. In these women, supplementation with 1,000 IU of vitamin D3 per day over a 9-month period increased the mean serum 25(OH)D level to 27.5 ng/ml and reduced risk of falling by nearly a half. A meta-analysis of vitamin D and falls trials concurred that trials in populations with low 25(OH)D levels showed a positive effect of supplementation whereas those in replete populations were null [205]. Additional trials in adults with low 25(OH)D levels are needed to confirm this finding.

Consensus Statements:

For any area of medical research, observational and association studies often provide the first evidence that a subject needs to be investigated in depth. There are several types of studies in this category: ecological, cross-sectional, case-control, and cohort studies [210]. The latter can have either an extended longitudinal study design. Ecological and cross-sectional studies often inform cohort studies and RCTs. They may be large, and if consistent across many varying populations can provide important information. In addition, they may provide essential guidance in designing more definitive studies. Association studies are often able to include much larger populations than RCTs. They can also be analyzed by meta-analysis to provide hypotheses for RCTs. As in all other areas of medical research, careful review of observational and association studies will improve RCT design.

Many observational and association studies of vitamin D have led to hypotheses that vitamin D exerts effects beyond bone and mineral metabolism. For example, low serum concentrations of 25(OH)D were found to be associated with a higher incidence of cardiovascular disease [211, 212], in addition to the known associations of serum lipids, smoking, diabetes mellitus, obesity, and high blood pressure [213]. Several plausible potential mechanisms have also been identified in laboratory studies. Association studies help in RCT design by determining which other potential independent variables that must be accounted for in the trial.

A randomized controlled clinical trial is defined as a prospective study comparing the effect (i.e. benefit-to-risk profile) of one intervention against another (either control or active), randomly assigned in humans [214]. Meta-analyses of well performed RCTs are at the top of the hierarchy of research evidence to advance clinical care. RCTs share many components with other study designs (e.g. need for an underlying hypothesis, selection of a population of interest, comparison of well-defined interventions, definition of outcomes) but, when well designed, they differ from other study designs in a single important element: a low probability for bias.

When "comparing" interventions, clinical trials should respect the principle of equipoise, which is a defined as a true state of uncertainty among the expert medical community and the researchers regarding the comparative efficacy-safety balance of each arm in a trial. Although equipoise is an elegant principle, it is difficult to implement in practice for a number of reasons, the main one being persistent researcher bias [215].

A distinct advantage of vitamin D trials, unlike other interventions with nutrients, is that serum 25(OH)D concentration can be measured to assess the effect of and adherence to the intervention (vitamin D supplementation). Importantly, the serum 25(OH)D concentration in RCTs is primarily the direct result of the study intervention and not a marker of health or disease, as in the observational studies with vitamin D. Therefore, the trial result (whether positive or negative) associated with a higher vs lower serum 25(OH)D concentration can be attributed to the trial intervention, emphasizing the importance of standardized measurements of 25(OH)D in RCTs.

The main limitation of a clinical trial is that it is designed, powered and conducted for a single primary outcome in a narrow population to test a very specific intervention. For example, in a recent study, monthly vitamin D3 supplementation of 100,000 IU for a median of 3.3 years did not prevent cancer [216]. That result does not necessarily mean that vitamin D3 supplementation over 5 or 10 years will not prevent cancer, as it may take longer for cancer to either develop and progress to a clinically apparent state.

A properly planned and executed RCT is a powerful experiment for assessing the effect-to-safety balance of an intervention and to guide clinical care. On the other hand, poorly designed, conducted, analyzed and reported trials can be misleading, potentially more than other study designs. Therefore, a successful trial is not one that only achieves a positive result, but one that reliably answers the primary question the trial was designed and conducted to answer. Finally, without supportive evidence, no single RCT (even a well conducted one) ought to be considered definitive and consistency among RCTs should be sought. Researchers and clinicians also need to balance RCT results with results from laboratory, animal and observational studies to establish a chain of causation.

Several factors can explain a lack of vitamin D efficacy on clinical outcomes in recent RCTs. Low doses as well as high doses may be inefficient, with a U-shape relationship existing between vitamin D doses and outcomes, for instance, fall risk or mortality [203]. A feature of vitamin D overdosing is the increased risk of fracture and falls observed with the yearly or monthly administration of large doses of vitamin D (corresponding however to a mean daily dose of 1,350 IU or 2,000 IU, respectively) [217]. These results provide important information for future clinical trial design. Additional secondary and exploratory outcomes should also be considered to further identify other potential effects of vitamin D.

The population included in the study is of major importance. A vitamin supplementation is more likely to produce a clinically significant effect in patients with hypovitaminosis D than in community dwelling healthy vitamin D sufficient individuals. Thus, vitamin D supplementation should be tested in vitamin D deficient subjects.

For fracture risk reduction, most positive trials have used a combination of calcium and vitamin D, emphasizing the importance of cofactors in vitamin D efficacy. The design of an ideal RCT with vitamin D should be adapted to the targeted disease and to the selected primary outcome. It could include a non-intermittent regimen with one or 2 moderate doses. The duration should be sufficient to record an adequate number of events, but limited to ensure optimal adherence [218]. Ideally, the population should be, or predicted to be, deficient in vitamin D. Controls may be either placebo or poorly effective low doses of vitamin D.

The number of subjects should be large enough so that the 95% confidence intervals are smaller than the expected effects. This implies also a full control of confounding factors and co-morbidities. Taken together, these various conditions suggest that an ideal RCT with vitamin D will be difficult in terms of patient recruitment, and expensive with, in addition, the risk of enrolling a population that is too selective. The more highly selective the population is, the greater uncertainly as to its general applicability. However, selection of a population expected to be moderately to severely deficient (serum 25(OH)D <30 nmol/L) would allow multiple outcomes to be studied in an RCT of vitamin D supplementation.

The VDR is found in essentially all cells of the body. The VDR ligand regulates the transcriptional activity of thousands of genes. Cellular and animal studies have documented the major impact vitamin D has through the active ligand 1,25(OH)2D and receptor VDR have on numerous biochemical and physiologic pathways and processes. Association studies in humans suggest that low serum 25(OH)D concentrations contribute to a large and growing list of medical conditions. However, RCTs testing the ability of vitamin D to exert beneficial effects on these medical conditions, other than the treatment of rickets/osteomalacia, have been remarkably unimpressive overall. Why? There are at least two possible explanations. The first is that the impressive data from cells and animals do not translate to the human condition. One might relegate this possibility to highly unlikely. A second explanation is that flaws in the design, conduct, analysis, and reporting of the RCT can bias the results achieved.

In 2005, the Cochrane Collaboration began a process to develop a new tool for assessing risk of bias in RCTs [219]. This tool has been broadly adapted with minor modifications in meta-analyses of a variety of vitamin D supplementation (and other) RCTs. As tabulated by Higgins et al., there are 6 major bias domains [219]. The first is selection bias. This domain includes an assessment of the methods involved in randomization of participants. Moreover, the size of the trial needs to be large enough to minimize differences between the groups and the power to robustly answer the question being posed. Equally important is whether the subjects selected to participate in the trial are appropriate for the question being asked. Expecting a benefit of vitamin D supplementation in subjects with normal serum vitamin D concentrations is probably unrealistic. The second is performance bias. This focuses on the blinding of participants and personnel managing the study. A risk in vitamin D RCTs is if participants sense they are receiving a placebo they begin taking or altering their intake of vitamin D and calcium, minimizing their difference with those in the active arm of the study. Detection bias is the third domain, and includes the assessment of measures used to blind those personnel responsible for evaluating the outcome from knowledge of the intervention. That said, determining the serum 25(OH)D concentration before and after the intervention in a vitamin D RCT is critical for determining not only compliance, but whether a response to the intervention can reasonably be expected. However, such testing will dramatically increase the expense of an RCT and, therefore, has the potential of limiting study size and power. Attrition bias is the fourth domain and includes how incomplete data are handled. In a lengthy trial, drop outs and decreased compliance complicate the analysis. Should subjects assigned to vitamin D supplementation, but who stopped or limited their vitamin D doses be considered as part of the active arm of the study, even if their 25(OH)D levels do not increase? Reporting bias is the fifth domain. The risk here is that of selective reporting. A trial may not reach its primary endpoint, but with data mining, other results may be found to be significant. Can results from a vitamin D RCT for treatment of osteoporosis be used to determine whether there was a reduction in cancer incidence? Finally, there is a category listed as other biases. This could include sources of funding. For vitamin D RCTs, this could also include design issues such as duration of the trial, doses expected to be adequate to achieve the desired effect, and the appropriateness of the dosing intervals.

A moderate to high risk of bias confounds many of the RCTs that we rely on for determining the efficacy of vitamin D supplementation on a number of medical conditions that animal and human association studies suggest could benefit from such supplementation. The risk of these biases impacting the interpretation of the reported results is compounded by trials in which participants are: 1) not preselected for vitamin D deficiency; 2) adherence is poorly monitored and/or not adjusted for in the analysis; 3) the dose of the vitamin D provided is not sufficient to achieve a significant increase in serum 25(OH)D concentrations; 4) the dosing intervals are too infrequent to provide a steady level of serum 25(OH)D concentrations; and 5) the effect of the administered dose on 25OHD is not determined and/or adjusted to ascertain that an effective level of 25(OH)D has been achieved. The degree to which these concerns have resulted in the large number of negative or minimally effective results with vitamin D supplementation for extra-musculoskeletal conditions is unclear. Future investigation using stricter criteria for RCT design, conduct, analysis, and reporting are necessary to address these concerns.

The ideal biochemical target for replacing vitamin D can be identified by interpolating recommended dietary allowances for vitamin D with evidence from pre-existing randomised controlled trials to balance concerns regarding efficacy and safety.

The IOM has set the recommended dietary allowances for vitamin D at 600 IU/day for adults aged 50-70 years and 800 IU/day for those aged >70 years, which would correspond to a serum 25(OH)D level of 50 nmol/L, sufficient to maintain bone health in 97.5% of North Americans [35, 48]. These are population-based recommendations, however, whereas data from meta­analyses of randomised controlled trials suggest that additional and optimal benefits may accrue at levels of 60-75 nmol/L and 90-100 nmol/L for musculoskeletal and non-musculoskeletal outcomes, respectively. For example, using a daily dose of vitamin D, falls are reduced at serum 25(OH)D of about 60 nmol/L, while non-vertebral fractures are reduced at levels 75 nmol/L. For multiple sclerosis, as an example of non­musculoskeletal outcomes, it seems a higher biochemical target of at least 100 nmol/L is needed for prevention. For a typical older adult, achieving a serum 25(OH)D level of 75 nmol/L requires a daily vitamin D dose of at least 800-1,000 IU. The rise in serum 25(OH)D is also slower in individuals with higher baseline intake.

These considerations have been taken into account when planning currently ongoing or recently completed large population-based RCTs of vitamin D. In planning the Vitamin D and Omega-3 trial (VITAL) comprising 25,871 older men (aged >50 years) and women (aged >55 years), given a daily dose of 2,000 IU or placebo. The mean serum 25(OH)D concentration level at baseline was 77 nmol/L and 12.7% had levels below 50 nmol/L. In participants with repeat measurements after 1 year, mean serum 25(OH)D concentrations increased by 40 nmol/L at 1 year [223]. The primary study outcomes of cancer and major cardiovascular events were both negative after a median 5.3 years of follow-up. Secondary end-points for death from cancer, breast cancer, prostate cancer, colorectal cancer; the expanded composite end-point of major cardiovascular events plus coronary revascularization, myocardial infarction, stroke, and death from cardiovascular causes; death from any cause were also all negative. No excess risks of hypercalcemia or other adverse events occurred in the vitamin D group. However, the rate of death from cancer over time was significantly lower with vitamin D than with placebo (HR 0.79 [95% CI, 0.63 to 0.99], and HR 0.75 [95% CI, 0.59 to 0.96], respectively). In analyses restricted to deaths from cancer in patients with adjudication of the cause of death, HRs were 0.72 (95% CI, 0.52 to 1.00) over the total follow-up period, and 0.63 (95% CI, 0.43 to 0.92) when the first 2 years were excluded.

In the ongoing Australian D-Health trial, 21,315 older adults have been randomised to receive either 5 years of oral vitamin D (60,000 IU/month) or placebo and will be followed for a total of 10 years for total mortality, as a primary outcome, with multiple secondary outcomes including total cancer and colorectal cancer incidence, falls, fractures and infections [224].

The first of the vitamin D mega-trials to publish is the Vitamin D Assessment (ViDA) Study carried out in Auckland, New Zealand, 2010 to 2015. Like the VIDAL pilot trial in the UK [225] ViDA [226] has employed higher monthly doses of 100,000 IU compared with placebo (with an initial bolus of 200,000 IU in ViDA). The study enrolled 5,110 adults, aged 50-84 years, recruited mainly from family practices with vitamin D3 (2.5 mg or 100,000 IU) or placebo softgel oral capsules, mailed monthly to participants' homes. Outcomes were monitored through routinely collected health data and self-completed monthly questionnaires. The mean baseline serum 25(OH)D concentration was 66 nmol/L and the mean post-treatment serum 25(OH)D concentration obtained in ViDA was 116 nmol/L.

The results showed no beneficial effect of vitamin D supplementation on incidence of cardiovascular disease [226], falls [227], non-vertebral fractures [227] and all cancer [216]. However, beneficial effects from vitamin D supplementation were seen: for persistence with taking statins in participants on long-term statin therapy [228]; and also in bone mineral density [229], and arterial function (central blood pressure) [230], particularly in participants with low serum 25(OH)D concentrations. Beneficial effects were also seen on lung function (forced expiratory volume in 1 second) among ever smokers, and patients with Chronic Obstructive Pulmonary Disease (COPD) or asthma (especially if vitamin D deficient) [231]. The latter findings are consistent with several previous studies. The beneficial effect seen on statin persistence [228] is preliminary and needs to be confirmed by other studies.

The lack of effect could be due to the use of monthly, rather than daily or weekly supplementation, insufficient participants with vitamin D deficiency, lack of co-supplementary calcium or too short a follow-up period for chronic disease outcomes. However, the ViDA study did show that the beneficial effects for some outcomes such as BMD [229], FEV1 [231] and arterial function [230] are more pronounced in subjects with serum 25(OH)D concentrations of <50 nmol/L. These findings are consistent with several previous studies, including VITAL and D2d, which collectively suggest that vitamin D supplementation may only be beneficial in people who are deficient [232].

The European Vitamin D3 - Omega3 - Home Exercise - Healthy Ageing and Longevity Trial (DO-HEALTH) enrolled 2,157 older men and women aged >70 years and treated them with Vitamin D 2,000 IU per day, marine omega-3 fatty acid 1g/d and exercise in a 2x2x2 factorial design for 3 years with multiple study endpoints including functional and cognitive decline, falls and fractures [233].

The Finnish FIND study randomised 2,495 older men and women to either 3,200 IU or 1,600 IU vitamin D per day for 5 years with cancer and cardiovascular disease as primary outcomes [234].

Finally, the International Polycap Study 3 (TIPS-3) will enroll 5,000 older men and women at high risk of cardiovascular disease and randomize to 60,000 IU vitamin D per month versus a polycap containing thiazide, atenolol, ramipril, simvastatin or placebo in a 2x2x2 factorial design for 5 years with 5 years of follow-up, having major adverse cardiovascular events (MACE) as the primary endpoint [235].

In a previous trial of vitamin D in vitamin D-deficient adults at risk of diabetes, a median daily dose of 4,000 IU (range 2,000 IU - 6,000 IU) was required to achieve a target serum 25(OH)D level of >75 nmol/L, because all trial participants were either overweight or obese [236]. In the ongoing Vitamin D and Type 2 Diabetes Study (D2d) in USA, in 2,423 individuals aged >25 years with risk factors for diabetes mellitus, a daily dose of 4,000 IU has been chosen with incident diabetes over 3 years being the primary outcome [237].

In conclusion, the equivalent daily doses in the large ongoing or recently completed vitamin DRCTs range from 2,000 - 4,000 IU and all aim for a target serum 25(OH)D concentration 90-100 nmol/L as non-musculoskeletal outcomes are primary study outcomes.

The first indication that vitamin D could increase the risk of fracture was reported in a trial that involved supplementation with the high dose of 300,000 IU of vitamin D2 versus placebo intra­muscularly once per year for 3 years in 9,440 adults aged 75 years and older [239]. In that pragmatic trial, vitamin D had no effect on non-vertebral fractures, but it significantly increased the risk of hip fracture. This study was followed by another in which annual dosing with 500,000 IU of vitamin D3 significantly increased the number of falls and fractures in women aged 70 years and older [217]. Increased risk of falling has subsequently been observed in a trial that employed monthly dosing with 60,000 IU of vitamin D3 [198]. The comparator group in this trial received 24,000 IU per month. Two trials have tested the effect of 100,000 IU of vitamin D per month on fall risk, one in community-dwelling elders [227] and the other in nursing home residents [240]. This dose had no effect on fall risk in the community-dwelling elders but it doubled the risk of falling in the nursing home residents. The initial mean serum 25(OH)D concentrations in the two studies were similar, 60.9 and 57.4 nmol/L, respectively.

Vitamin D in combination with supplemental calcium can, under selected circumstances, increase the risk of kidney stones. In the 7-year Women's Health Initiative (WHI), daily treatment with 400 IU of vitamin D3 plus 1,000 mg of calcium significantly increased risk of kidney stones by 17% when compared with the placebo [241]. The increased risk did not emerge until the 5^th year of supplementation [242]. WHI participants were allowed to continue to take their personal calcium supplements and as a result, initial mean calcium intake was over 1,100 mg per day. With supplementation, it exceeded 2 grams per day. Increased stone risk has not been seen in other trials, possibly because the other trials have been of shorter duration and have not allowed personal calcium or vitamin D supplement use. In a recent 4-year trial testing the effect of 2,000 IU of vitaminD3 plus 1,500 mg of calcium daily for 4 years on cancer risk, there were 16 kidney stones in the supplemented group and 10 in the placebo group [128].

With respect to the risk of cardiovascular disease, the large ViDA study administered 100,000 IU of vitamin D3 or placebo monthly for 3.3 years to adults age 50-84 years and reported no effect on cardiovascular disease events (primary outcome) [226]. Observational studies have noted a reverse J-shaped association of serum 25(OH)D with cardiovascular disease mortality [243]. However, a meta-analysis of 24 RCTs concluded that there was no effect of vitamin D alone on mortality but that vitamin D with calcium reduced mortality in the elderly [244].

In conclusion, high intermittent doses of vitamin D have significant potential to increase the risk of falls and fractures and are therefore an undesirable dosing regimen. Long-term use of vitamin D in combination with substantial doses of calcium (1,000 to 1,500 mg per day) has increased risk of kidney stones in participants with relatively high usual calcium intake. Prevailing evidence indicates that vitamin D supplementation does not significantly alter risk of cardiovascular disease events. Vitamin D supplementation has not been demonstrated to alter mortality and vitamin D together with calcium may reduce mortality.

Despite our incomplete knowledge of the role of vitamin D in many target tissues, we know that serum 25(OH)D concentrations <50 nmol/L are likely to have adverse effects on health and that this affects one-quarter of the world’s population. This is a global health problem that needs to be corrected. The ideal RCTs for evaluating health benefits of vitamin D supplementation should have the following charachteristics [245‐248]: