Review of Vitamin D (including free, frequency, injection, how much.) – Sept 2013

Value and limitations of assessing vitamin D nutritional status and advised levels of vitamin D supplementation

European Journal of Endocrinology (2013) 169 R59-R69
Elisabetta Romagnoli, Jessica Pepe, Sara Piemonte, Cristiana Cipriani and Salvatore Minisola
Department of Internal Medicine and Medical Disciplines, University of Rome 'Sapienza', Viale del Policlinico 155, 00161 Rome, Italy
(Correspondence should be addressed to S Minisola; Email: salvatore.minisola at fastwebnet.it)

The growing attention to the role of vitamin D in skeletal and extra-skeletal diseases over the last decade induced an increased demand for vitamin D determination as well as a dramatic rise of sales of vitamin D supplement. However, several critical points in this field remain to be clarified. We lack a clear consensus about the definition of vitamin D deficiency, insufficiency, and sufficiency. The identification of different thresholds defining vitamin D status has relevant implications in clinical practice. In fact, the worldwide prevalence of low vitamin D status is highly varying according to the level of 25(OH)D utilized to define sufficiency. Therefore, the assessment of 25-hydroxyvitamin D levels may have a critical role, but a number of different technical problems associated with its determination may interfere in interpreting the results. The hydrophobic nature of vitamin D and the tight binding to its carrier (vitamin D binding protein), the different forms circulating in blood, and the issue of standardization are among the most important factors influencing the measurement of this metabolite. Another controversial point relies on the conflicting guidance on prevention and treatment of vitamin D deficiency endorsed by different medical and scientific communities. In particular, uncertainty exists about how to replete vitamin D stores, how to maintain normal 25(OH)D levels after repletion, which form of vitamin D is preferable for supplementation, and which route of administration and dosing regimens are advisable. Finally, concerns have been raised regarding vitamin D toxicity and its adverse effects.

Introduction

There has been a growing interest in vitamin D during the last decades, which has boosted an increasing number of scientific papers on this topic. This interest, also shared by the lay community, mainly derives from the recognized effect of vitamin D on mineral metabolism and neuromuscular function (1, 2) and the purported effect on other aspects of health: cardiovascular (3, 4, 5), endocrine (6, 7), metabolic (8), neurological (9, 10), neoplastic (11), articular (12), immunological (13, 14), etc. Furthermore, vitamin D has also been linked to mortality (15,16). The logical consequence of this surge of attention has been an increased demand for the determination of serum 25(OH)D levels (the best available index of vitamin D nutritional status) with substantial associated costs, in order to prove that insufficiency or deficiency of vitamin D was the causative factor of that particular disease and, vice versa, when the subject was repleted with vitamin D, he/she was protected or could be considered at lower risk.
Vitamin D is mainly derived from sun light exposure of the skin (17), only one-fifth being introduced by dietary sources from animal (cholecalciferol-D3) or plant (ergocalciferol-D2) origin. In order to be fully active, both ergocalciferol and cholecalciferol undergo 25-hydroxylation in the liver generating 25(OH)D2 and 25(OH)D3. This is the major rate-limiting step primarily dependent on the parent compound and therefore explaining the well-known seasonal variation of 25(OH)D (18). In normal subjects, the kidney adds an hydroxyl group in position 1 giving rise to the final metabolites 1,25(OH)2D2 and 1,25(OH)2D3. A reduction in serum calcium, phosphorus, or fibroblast growth factor 23 (FGF23) and an increase in parathyroid hormone (PTH) stimulate the activity of CYP2 7B1 hydroxylase. In this context, it is important to note that opposite changes (i.e. an increase in serum calcium, phosphorus, and FGF23 and a reduction in PTH) determine a conversion of 25(OH)D toward the production of 24,25(OH)2D. The possibility of producing another metabolite by inducing hydroxylation in position 26 (25,26(OH)2D) exists. The physiological role of these last two metabolites is still an object of debate (19).
Vitamin D status is defined by the measurement of 25(OH)D; this term refers to both circulating forms (25(OH)D2 and 25(OH)D3) of the vitamin. There are a number of reasons why the concentration of total 1,25(OH)2D cannot be utilized as a marker of vitamin D status; this is because of its short half-life (4-15 h vs 21-30 days of 25(OH)D), low concentrations of the final metabolite (picomole vs nanomole), and owing to the fact that a very small amount of 25(OH)D can be converted to 1,25(OH)2D, thus giving the false idea of sufficiency. Only when 25(OH)D falls below 4 ng/ml (corresponding to 10 nmol/l, being 1 ng/ml = 2.5 nmol/l), there is a concomitant decrease in 1,25(OH)2D(19).

Measurement of 25(OH)D

The diagnosis of hypovitaminosis D (either deficiency or insufficiency) is therefore based on the current concentration and measurement of total 25(OH)D. However, there are a number of technical problems that should be born in mind in order not to misinterpret the results.
There are at least three major reasons impeding the achievement of a robust result; these are represented by the hydrophobic nature of the compound with the tight binding to its carrier (vitamin D binding protein (DBP)), the different forms circulating in blood, and the issue of standardization (Fig. 1).
As 25(OH)D is a lipophilic substance tightly linked to DBP, this generates some technical problems. Furthermore, endogenous lipids may affect binding and chromatographic separation, as they co-extract from plasma and serum. An important preventive measure to be adopted is avoiding sunlight exposure of the sample because this may induce degradation ofthe vitamin; the latter also applies to the standard employed in some assays. In contrast, the 25(OH)D is a very stable metabolite; multiple freeze and thaw cycles have no significant effect on determination of 25(OH)D in serum (20, 21). Indeed, in one of the most recent papers addressing the problem of the optimal threshold for defining vitamin D status, the authors performed the measurement of 25(OH)D in a blood sample taken at autopsy; they stated that, unlike PTH and calcium, 25(OH)D was found to be stable in various experiments for at least 10 days postmortem (22).
Figure 1

Factors influencing serum levels of total 25(OH)D and its partition in blood. *24,25(OH)2D, 3-epimer-25(OH)D, isobars are recognized by some assays.

As previously stated, total circulating 25(OH)D is the sum of two metabolites, 25(OH)D2 and 25(OH)D3. However, not all the immunoassays employed in clinical practice are able to detect 25(OH)D2. Cavalier et al. (23) were one of first to enlighten this problem; indeed, they demonstrated that after vitamin D2 administration, contrary to what would have been expected, there was no increase in total serum 25(OH)D with one of the methods employed. This finding has obvious clinical implications in subjects treated with vitamin D2 or in countries (i.e. USA) in which vitamin D2 is the only FDA-approved product (24). This methodological problem, possibly related to a stronger affinity of the DBP for 25(OH)D2 (25), poses an individual treated with D2 at risk of vitamin D intoxication, because with some assays he/she will unlikely reach the 'laboratory' sufficiency. Therefore, according to the authorities in the field (19, 26), the ideal method of measurement should equally detect both metabolites. Isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) is currently considered the referent method for 25(OH)D assay because it measures 25(OH)D2 and 25(OH)D3. However, HPLC may also be utilized, and according to Cavaliers' (23) and our own data (27, 28), the Diasorin RIA is endowed with these characteristics.
Some other metabolites may be the origin of spurious results. Among them, it is worthwhile to mention 24,25(OH)2D, which may represent up to 10-15% of the total quantity of 25(OH)D. Antibody-based methods, particularly those involving no chromatographic steps, cannot resolve 24,25(OH)2D and include this metabolite in the estimation of total 25(OH)D. Some commercial kits offer corrections for this metabolite but such correction appears to be inaccurate at high or low values. Recently, there has been new interest in the assay of 24,25(OH)2D3 owing to some findings demonstrating, for example, that the enzyme 24-hydroxylase (CYP24A1) is stimulated by FGF23 (29); that idiopathic infantile hypercalcemia may in part be derived by loss-of-function mutations in CYP24A1, so that the levels of this metabolite are undetectable (30); and that CYP24A1 defects in adults are associated with nephrolithiasis or nephrocalcinosis (31). Furthermore, genome-wide association studies have demonstrated that CYP24A1 variation is one of the four genetic determinants identified so far causing variability of serum 25(OH)D (32); therefore, the levels of 25(OH)D may also reflect fast and slow metabolizers with corresponding high or low serum 24,25(OH)2D levels.
There are two other substances that can be the cause of spurious results; the first one is the 3-epi-25(OH)D epimer which is a related molecule present in varying concentrations in normal subjects (33) that may interfere with the results obtained by LC-MS/MS. Another possible interference could derive from isobars, even though more detailed investigations are needed concerning these substances (19, 34). Epimers and isobars are compounds with the same molecular weight as that of vitamin D metabolites forming the same mass to charge parent and product ion pairs upon ionization.
One of the most important problems in this field is represented by the great variability in the results obtained among laboratories that utilize different methods, as also recently underscored (35). This is an old problem (36), partly overcome in recent years, mainly derived by the lack of a reference standard; before the adoption of such a standard, there was also a great variability when comparing three different laboratories employing what is now considered the gold standard of measurement, i.e. the LC-MS/MS (37). The absence of certified reference material for 25(OH)D is the most important factor determining the imprecision in identifying individuals with vitamin D levels below the optimal threshold, anyway defined; this often leads to the perception that an individual was classified as sufficient or insufficient based on the laboratory used for the determination. This has obvious important clinical implications, particularly in redefining worldwide vitamin D status (38), as demonstrated in a recent paper by Perna et al. (39). The National Institute of Standards and Technology (NIST) has developed a standard reference material (SRM 972) in order to solve this problem. The SRM consists of four pools of serum, each with varying levels of vitamin D metabolites. Chromatographic resolution of the 3-epimer of 25(OH)D3 proved to be essential for accurate measurement of the vitamin D metabolites present in these serum samples (40). The importance of the standardization process is demonstrated by the success story of serum total cholesterol (41). Presently, there are several ways for participation in the vitamin D Standardization Program (VDSP). Among them, the NIST-NIH Vitamin D
Metabolites Quality Assurance Program (http://www. nist.gov/mml/csd/vitdqap.cfm), the DEQAS program, the VDSP's CDC Standardization-Certification Program, and finally the possibility of collaborating with VDSP to standardize 25(OH)D made sometime in the past as part of studies that have been completed. Standardizing values measured in the past require re-measuring total 25(OH)D concentration in a statistically designed subsample of stored sera (~100 samples) from the study by a laboratory that has been standardized to the NIST reference measurement procedure (41).

Vitamin D binding protein

DBP is the main serum carrier of vitamin D metabolites (albumin is a lower affinity binder), whose published normal reference range is 30-60 mg/dl (42). In physiological conditions, about 83% of total 25(OH)D in the circulation is bound to DBP (42, 43). The term bioavailable 25(OH)D refers to the readily available form of circulating vitamin D, that is free 25(OH)D combined with albumin-bound 25(OH)D.
Recently, the interest in DBP has considerably increased. DBP circulates in three major polymorphic forms, thus producing six allelic combinations occurring at different frequencies among ethnic populations (44). The different allele forms of DBP circulate at varying concentrations and possess different binding affinities for 25(OH)D and 1,25(OH)2D; therefore, both these variables have the potential to influence bioavailability of vitamin D (44). These data are in accordance with the recent genome-wide association studies showing that lower affinity forms of DBP are associated with lower circulating levels of 25(OH)D so that the affinity of the binding may regulate both the total and free 25(OH)D levels (33, 45) (Fig. 2). In this context, it is important to note that a recent study has demonstrated the association between bone mineral density (BMD) and levels of free 25(OH)D but not total circulating values of the vitamin (46). Along these lines, a recent longitudinal study showed that the known associations of low 25(OH)D concentrations with clinical outcome are related to common genetic differences in the vitamin D receptor (47).

Vitamin D and the search for a threshold

Figure 2

A theoretical approach to the interrelationships among total, free vitamin D and binding affinity of vitamin D binding protein (DBP). Depending on the isoform present in serum, the active 25(oH)D fraction (that is, the free fraction) may be elevated or reduced despite corresponding reduced or elevated total values of the vitamin. Different DBP turnover rates for the genetic variants may also have a role.

The definition of vitamin D deficiency, insufficiency, and sufficiency is currently challenging as an overall consensus is still lacking (48, 49, 50). It represents a crucial issue, as the identification of different thresholds defining vitamin D status has varying implications in clinical practice. First, the worldwide prevalence of low vitamin D status is highly varying according to the level of 25(OH)D utilized to define sufficiency. Consequently, the choice to initiate vitamin D supplementation may change, as well as the goals of therapy, the dosing strategy, and the decision about who should be screened, if necessary, and how often (51).
In recent years, a number of position statements and clinical practice guidelines have been published to define the optimal vitamin D status and the health outcomes associated with its alteration (52, 53). Many different recommendations on dietary intakes needed to reach and maintain sufficient 25(OH)D levels have been
proposed as well (54, 55, 56, 57, 58, 59).
In this context, the publication of the two most authoritative reports on these issues (one released from the Institute of Medicine (IOM) committee's 2011 report on dietary reference intakes for calcium and vitamin D and the other from the Endocrine Society clinical practice guideline for the evaluation, treatment, and prevention of vitamin D deficiency) (58, 59) has lead to confusion among clinicians, researchers, and the public because of the disagreement in data interpretation. The conclusions of the two reports indeed differ considerably. The US Endocrine Society (ES) reported a 25(OH)D level <20ng/ml (50 nmol/l) as the 'cut off' to define vitamin D deficiency, a 25(OH)D level between 21 and 29 ng/ml (52.5 and 72.5 nmol/l) to define vitamin D insufficiency, and a 25(OH)D level more than 30 ng/ml (75 nmol/l) as the optimal level. In contrast, the IOM concluded that 25(OH)D levels above 20 ng/ml are needed for good bone health for almost all the individuals (97.5% of the population), while a level of 16 ng/ml (40 nmol/l) meets the need of approximately half the population. According to the IOM, higher levels of 25(OH)D have not been consistently shown to confer greater benefits, in turn challenging the concept that 'more is better'. The controversy has been fueled by several factors that should be taken into account in interpreting the results of the current literature. These include: the difficulty to distinguish the sole effect of vitamin D as the majority of intervention trials co-administered calcium; the difficulty to exactly measure the relative contribution of sunlight exposure, food fortification, and multivitamins intake; the lack of randomized controlled trials assessing the effect of vitamin D supplementation on health outcomes other than bone; and the complexity to compare studies utilizing different 25(OH)D assays (24, 60, 61, 62, 63). On the other hand, it should be emphasized that the two reports are intended for different populations (the general population for the IOM report and the population at risk for deficiency for the ES), and this could partly explain the controversy surrounding their respective recommendations.
The primary health outcomes of vitamin D nutrition utilized to define vitamin D sufficiency are those related to skeletal health. Actually, maximal intestinal calcium absorption, serum PTH suppression (52, 53), reduced risk of falling, prevention of fractures, increase in BMD, and reduced histomorphometric findings of osteomalacia from bone biopsy are the most important parameters considered in both reports to identify the optimal vitamin D status (58, 59, 64, 65, 66).
Table 1 briefly summarizes the different conclusions about these skeletal outcomes reached by the two professional organizations. Concerning the other possible benefits of vitamin D, both reports concluded that existing data are not sufficient to support the recommendation of vitamin D supplementation to reduce the risk of extra-skeletal acute and/or chronic diseases (58, 59).

Vitamin D and supplementation: general considerations

The controversy between the IOM and the ES on the definition of 'sufficiency' and the different goals of supplementation and treatment generated very different recommendations about vitamin D intakes. The IOM concluded that children aged 0-1 year require 400 IU/daily vitamin D (corresponding to 10 mg/daily vitamin D, being 1 ug vitamin D = 40 I U), all other children and adults up to the age of 70 years require 600 IU/day (15 ug/day) and adults over the age of 70 years need 800 IU/day (20 ug/day). On the contrary, the ES recommended a dose of vitamin D ranging from 400 to 1000 IU/day (10-25 mg/day) for children aged 0-1 year, 600-1000 IU/day (15-25 mg/day) for children aged more than 1 year, and 1500-2000 IU/day (37.5-50 mg/day) for adults aged 18 years or more (9, 10). Moreover, the ES also recognized that obese children and adults may require as much as two to three times the recommended dose due to the influence of body fat on vitamin D storage and metabolism (67).
The tolerable upper intake for those aged 9 years and older was set at 4000 IU/day (100 mg/day) by both the reports; however, the ES stated that larger doses may be needed to correct vitamin D deficiency in certain clinical situations (for example, 10 000 IU/day (250 mg/day) for adults aged >18 years). Also, the IOM recognized that such an intake is not associated with intoxication. Finally, both the IOM and the ES recommend that either vitamin D2 or vitamin D3 could be used as they have the same efficacy to raise and maintain circulating 25(OH)D levels (58, 59).

Vitamin D and the supplementation: the discussion

Table 1

The position of the IOM and the US Endocrine Society (ES) in the definition of vitamin D sufficiency based on different skeletal outcomes.
Skeletal outcome
Intestinal calcium absorption
Evidence of osteomalacia by histomorphometry from bone biopsy
Relationship between serum 25(OH)D and serum PTH levels
Fracture risk reduction
Fall risk reduction
Increase in BMD
IOM
A threshold for normal calcium absorption occurs at a serum level of 25(OH)D of 5-10 ng/ml (102, 103)
Only 1% of patients with 25(OH)D levels above 20 ng/ml have osteomalacia (23)
No evidence for a plateau of serum PTH levels or a plateau for 25(OH)D levels between 10 and 50 ng/ml; however, PTH levels can vary significantly by sex, age, time of day, kidney function, and dietary calcium (105, 106)
Vitamin D at 800 Ul/day provides some benefit in fracture risk reduction in elderly but only when calcium supplementation was added to the treatment (108, 109, 110)
Vitamin D supplementation does not prevent falls. The reanalysis of data presented by the US ES indicates that there is no significant dose-response relationship between the risk of fall and the achieved 25(OH)D levels (63, 113)
Intervention studies showed little increase in BMD in vitamin-D-replete participants. Moreover, little or no additional benefit in BMD is observed with serum 25(OH)D levels above 20 ng/ml (116,117,118)
US ES
Calcium absorption is optimized at serum 25(OH)D level of 30 ng/ml (104)
No evidence of osteomalacia in all patients with 25(OH)D more than 30 ng/ml (8.5% of patients with 25(OH)D levels above 20 ng/ml have osteomalacia) (23)
PTH begins to plateau in adults who have 25(OH)D levels between 30 and 40 ng/ml (1, 107)
Antifracture efficacy of vitamin D started at 25(OH)D levels of at least 30 ng/ml.
This level is reached only in trials that gave at least 800 Ul/day vitamin D3 (111, 112)
Vitamin D supplementation at doses at least 800 UI/day reduces the risk of falls, particularly in elderly with vitamin deficiency at baseline. A gain in lower extremity strength or function and an improvement in body sway are also observed (115, 117)
Higher hip BMD is associated with high serum 25(OH)D levels throughout the reference range of 9-37 ng/ml (119, 120)

Since the publication of these two differing recommendations, a lively debate ensued among clinicians and researchers on several controversial points.
In particular, uncertainty was raised about the following:

• i) how to replete vitamin D stores;
• ii) how to maintain normal 25(OH)D levels after repletion;
• iii) which form of vitamin D is preferable for supplementation;
• iv) which route of administration and which dosing regimens are advisable; and
• v) vitamin D toxicity and adverse effects.

Achieving and maintaining vitamin D sufficiency

The optimal dosage to reach sufficiency remains poorly defined. In general, according to a rule of thumb, for every 100 IU (2.5 mg) vitamin D taken, 25(OH)D levels increase to about 1 ng/ml, but with a huge inter-individual variability.
Several factors may account for such a variability: the initial 25(OH)D concentration, patient's weight, adequacy of the dose according to compliance, the type of vitamin D administered (D2 or D3), renal function, and genetic factors. The variability in absorption, the inaccuracy of 25(OH)D assessment, as well as unknown factors also probably contribute to the variability of the dose-response relationship (68, 69, 70).
Controversy also exists on whether supplementation should be given daily or intermittently (e.g. weekly, monthly, quarterly, or once a year). It has been shown that circulating levels of 25(OH)D increase similarly when oral vitamin D is given daily, weekly, or monthly, provided that the total amount is identical. However, it must be recognized that a universal supplementation guideline does not exist, most likely the result of great disparity among countries in the availability of vitamin D supplements (71).
Another crucial point is that the immediate aim of treatment should be quick normalization of 25(OH)D levels, as well as vitamin D stores. This quick 'correction' can be accomplished with an initial period of high-dose vitamin D. An intermittent high-dose therapy (the so-called 'Stoss' therapy) is an interesting option to avoid non-adherence to treatment, although a regimen of regular low dose is a reasonable alternative. Studies comparing these two different regimens actually reported inconsistent results, and both high dose (dosing interval < 2 months) and more regular low dose seem to offer similar efficacy (72, 73, 74). The maximum safe bolus of vitamin D remains uncertain. A number of papers reported that a single oral dose of 300 000-600 000 IU D2 or D3 rapidly enhances serum 25(OH)D and reduces PTH levels in patients with deficiency (27, 28, 75). However, the study by Sanders et al. (76) showing that 500 000 IU oral dose of cholecalciferol increased the risk of falls and fractures among older women deserves attention. Another trial reported that 300 000 IU ergocalciferol given i.m. for 3 years to elderly people during fall season did increase fracture risk (77). No plausible biological explanation has been given for these results, whose interpretation remains merely speculative. However, these papers raise the possibility that infrequent high doses of vitamin D may be unsafe, probably because they induce large and rapid fluctuation in vitamin D status, thus counteracting any possible beneficial outcome. The debate is still open: the rate and magnitude of the increase in serum 25(OH)D levels may be critical, as well as at which time points 25(OH)D concentration should be measured after dosing. On the other hand, it is undeniable that, on a population basis, the utilization of intermittent large doses could aim to overcome the problem of compliance (78, 79, 80, 81).
Once vitamin D stores have been replete, a maintenance dose of 800-2000 IU/day (20-50 mg/day) should be recommended. In particular, long-term supplementation has to be encouraged in special groups that are at high-risk for deficiency. In this regard, many experts have questioned the IOM recommendations as, in the absence of sun exposure and dietary input, a daily dose of 600 IU (15 mg) vitamin D will not maintain blood 25(OH)D levels, even at 20 ng/ml (82, 83). Therefore, higher doses may be necessary to achieve an optimal vitamin D status. Indeed, published data demonstrate that among postmenopausal women, larger doses of between 800 and 2000 IU (20 and 50 mg) vitamin D daily, were not able to achieve sufficiency in all the participants (84, 85). Moreover, in a recent study, Cavalier et al. (86) reported that the administration of about 4000 IU/day (100 mg/day) of vitamin D3 in subjects with baseline serum 25(OH)D levels < 10 ng/ml was insufficient to achieve or maintain 30 ng/ml in a significant proportion of subjects. It is noteworthy that the administered dose was very close to the upper safety limit of 4000 IU/day defined by the IOM. We believe that high-risk populations, such as the elderly and institutionalized individuals, should receive a supplementation of higher-than-usually accepted doses to achieve the desired level. Recently, a panel of French experts published specific guidelines for vitamin D supplementation in nursing home residents. The panel agreed that all nursing home residents should be supplemented with a dose of at least 1000-2000 IU/day (25-50 mg/day) vitamin D3 given intermittently (e.g. weekly, monthly, quarterly, or once a year) to improve compliance and to reduce both daily poly-pharmacy and the burden for the nursing home personnel (87).
Comment by Vitamin D Life: quarterly and annually are far too infrequent

Vitamin D supplementation: which type?

Current evidence suggests that ergocalciferol has a considerably lower efficacy than cholecalciferol in raising circulating 25(OH)D levels. This difference between the two calciferols relates to several factors: the different affinity for the DBP and VDR, the different affinity as substrate for hepatic 25-hydroxylase, and a possible difference in the 24-hydroxylation rate. This last point deserves interest. In fact, the metabolism of vitamin D involves 24-hydroxylation in the kidney to form 1,24,25(OH)3D. This step is crucial as once 1,24,25(OH)3D2 has been formed, ergocalciferol has been deactivated and, therefore, is irretrievable. On the contrary, the 1,24,25(OH)3D3 still binds to VDR ( = 40% more than 1,25(OH)2D3) and must undergo additional side-chain oxidation to be biologically deactivated (88). This additional step gives a vast advantage and potential for cholecalciferol to remain biologically active and, thus, maintains vitamin D status. Available data also document the higher efficacy of cholecalciferol, regardless of the frequency of administration (small daily doses or in larger and more infrequent bolus) (27, 88, 89, 90, 91). The monthly administration of 500 mg oral 25(OH)D3 has been proposed as an alternative for vitamin D repletion, without any detrimental effect (92). Moreover, it has been recently demonstrated that 800 IU (20 mg) oral 25(OH)D3 per day resulted in a safe, immediate, and sustained increase in serum 25(OH)D levels in all participants compared with vitamin D3 (1 mg oral 25(OH)D3 increases 25(OH)D levels to about 4-5 nmol/l compared with the 1 nmol/l increase with 1 mg vitamin D3) (13). Taken together, these findings suggest that where available, calcidiol is an option for supplementation, particularly in specific clinical conditions such as advanced liver failure in which the 25-hepatic hydroxylation is impaired.

Vitamin D supplementation: i.m. or oral route of administration?

In many countries around the world, both cholecalciferol and ergocalciferol are available as oral or i.m. preparations. In general, oral administration is more physiological and leads to a rapid increase in serum 25(OH)D levels within 3 days (27). With i.m. injection, a gradual increase in serum 25(OH)D levels was observed, thus demonstrating a delayed serum 25(OH)D response (27). This phenomenon is probably due to the sequestration of vitamin D in the muscle and fat, where it is gradually released. It has been hypothesized that this pharmacokinetic profile potentially allows i.m. preparations to overcome the fluctuation of serum 25(OH)D levels following high oral bolus (81). However, this point has not been definitively clarified. On the other hand, i.m. preparations may have specific indications, in particular for intermittent (once- or twice-yearly) high-dose regimens. For example, in patients with short bowel syndrome, such an intermittent i.m. regimen is able to attain vitamin D sufficiency. Moreover, in children or in institutionalized elderly, the i.m. administration is effective in prevention of deficiency, also improving the long-term adherence to treatment (93).
Note by Vitamin D Life: Wrong: reference 93 found a 60 day delay in response, not a 180 or 360 day

Vitamin D supplementation and safety

Vitamin D intoxication is rather unusual. After intensive solar ultraviolet B (UBV) irradiation, the skin synthesis of vitamin D is self-regulated since inactive metabolites are produced. This explains why no reports described vitamin D intoxication from excessive sun exposure. Moreover, available data demonstrate that skin synthesis of up to 10 000 IU (250 mg) vitamin D daily is safe. The contribution of dietary intake is usually about 10-20%; hence, intoxication is nearly impossible from this source. Therefore, the potential harm of vitamin D may only come from the excessive ingestion of supplements (94, 95).
A number of studies linked the amount of vitamin D intake with the achieved serum 25(OH)D levels in order to establish a threshold for intoxication. It has been reported that there is no harm with an intake of 10 000 IU/day (250 mg/day) vitamin D, which corresponds to a serum 25(OH)D level of about 88 ng/ml (220 nmol/l) (96). Nevertheless, the IOM recently set the upper safe level of circulating 25(OH)D at about 50 ng/ml (125 nmol/l) on the basis of observational studies showing a U-shaped association between circulating 25(OH)D and some clinical outcomes (frailty, all-cause mortality, cancer, falls, and fractures) (97). Serum 25(OH)D levels beyond this limit are considered potentially harmful. However, this threshold seems to be very conservative, especially if we take into consideration many published studies showing that doses around 4000 IU/day (100 mg/day) vitamin D are safe, even in long-term treatment. The serum 25(OH)D level achieved in these studies was between 30 and 64 ng/ml (75 and 160 nmol/l), and it was not accompanied by any clinical sign of intoxication (95).

Vitamin D supplementation and the real world

Over the last decade, there has been growing attention to the role of vitamin D in various chronic diseases. However, the flourish claims in the media, the increasing scientific publications in peer-reviewed journals and the increasing information available on consumer health web sites had two relevant consequences: a substantial increase in laboratory testing for vitamin D and a dramatic rise of sales of vitamin D supplement. In the USA, many clinical laboratories have experienced increases in vitamin D testing of 100% or more in the last 5 years (98). The amount spent on vitamin D supplements in the USA had risen to tenfold in 10 years, from $40 million in 2001 to $425 million
in 2009 (99) and $600 million in 2011 (100). On the other hand, the scenario in Europe seems to be different. In fact, a recent survey carried out in southwest Scotland showed that 69% of patients in whom determination of serum 25(OH)D level was requested had vitamin D levels below 20 ng/ml (50 nmol/l) but only 61% of deficient patients were prescribed any form of vitamin D replacement therapy. Moreover, inadequate doses or inappropriate forms of therapy were frequently suggested (101). These findings highlight that the gap between expert recommendations and clinical practice could be partly explained by the conflicting guidance on definition, prevention, and treatment of vitamin D deficiency endorsed by different medical and scientific communities.

Conclusions and perspectives

Important advances have been made in the understanding of the metabolism, mode of action, and measurement of 25(OH)D. At the same time, the scientific community does not seem to find a consensus on the definition and treatment modalities of hypovitaminosis D. Future investigation should fill these gaps, focusing on accurate measurement of vitamin D without neglecting the possibility of determining the free fraction. The genetic variants regulating circulating 25(OH)D levels and how these traits can influence supplementation and treatment are definite areas of research.

References

• 1 Holick MF. Vitamin D deficiency. New England Journal of Medicine 2007 357 266-281. (doi:10.1056/NEJMra070553)
• 2 El-Hajj Fuleihan G, Nabulsi M, Tamin H, Maalouf J, Salamoun M, Khalife H, Choucair M, Arabi A & Vieth R. Effect of vitamin D replacement on musculoskeletal parameters in school children: a randomized controlled trial. Journal ofClinical Endocrinology and Metabolism 2006 91 405-412. (doi:10.1210/jc.2005-1436)
• 3 Anderson JL, May HT, Horne BD, Blair TL, Hall NL, Carlquist JF & Lappe DL. Relation of vitamin D deficiency to cardiovascular risk factors, disease status, and incident events in a general population. American Journal of Cardiology 2010 106 963-968. (doi:10.1016/j.amjcard.2010.05.02 7)
• 4 Pittas AG, Chung M, Trikalinos T, Mitri J, Brendel M, Patel K, Lichtenstein AH, Lau J & Balk EM. Vitamin D and cardiovascular outcomes. Annals of Internal Medicine 2010 152 307-314. (doi:10.7326/0003-4819-152-5-201003020-00009)
• 5 Wang TJ, Pencina MJ, Booth SL, Jacques PF, Ingelsson E, Lanier K, Benjamin EJ, D'Agostino RB, Wolf M & Vasan RS. Vitamin D deficiency and risk of cardiovascular diseases. Circulation 2008 117 503-511. (doi:10.1161/CIRCULATIONAHA.107.706127)
• 6 Grey A, Lucas J, Horne A, Gamble G, Davidson JS & Reid IA. Vitamin D repletion in patients with primary hyperpara-thyroidism and coexistent vitamin D insufficiency. Journal of Clinical Endocrinology and Metabolism 2005 90 2122-2126. (doi:10.1210/jc.2004-1772)
• 7 Carnevale V, Manfredi G, Romagnoli E, De Geronimo S, Paglia F, Pepe J, Scillitani A, D'Erasmo E & Minisola S. Vitamin D status in female patients with primary hyperparathyroidism: does it play a role in skeletal damage.? Clinical Endocrinology 2004 60 81-86. (doi:10.1111/j.1365-2265.2004.01946.x)
• 8 Dusso A, Gonzalez EA & Martin KJ. Vitamin D in chronic kidney disease. Best Practice & Research. Clinical Endocrinology & Metabolism 2011 25 647-655. (doi:10.1016/j.beem.2011. 05.005)
• 9 Miller JW. Vitamin D and cognitive function in older adults. Neurology 2010 74 613-615. (doi:10.1212/WNL.0b013e3181 c719a2)
• 10 Slinin Y, Paudel ML, Taylor BC, Fink HA, Ishani A, Canales MT, Yaffe K, Barrett-Connor E, Orwoll ES, Shikany JM et al. 25-Hydroxyvitamin D levels and cognitive performance and decline in elderly men. Neurology 2010 74 33-41. (doi:10.1212/ WNL.0b013e3181c7197b)
• 11 Freedman DM, Looker AC, Chang SC & Graubard BI. Prospective study of serum vitamin D and cancer mortality in the United States. Journal ofthe National Cancer Institute 2007 99 1594-1602. (doi:10.1093/jnci/djm204)
• 12 Chaganti RK, Parimi N, Cawthon P, Dam TL, Nevitt MC & Lane NE. Association of 25-hydroxyvitamin D with prevalent osteoarthritis of the hip in elderly men. Arthritis and Rheumatism 2010 62 511-514. (doi:10.1002/art.27241)
• 13 Bischoff-Ferrari HA, Dawson-Hughes B, Stocklin E, Sidelnikov E, Willett WC, Orav EJ, Stahelin HB, Wolfram S, Jetter A, Schwager J et al. Oral supplementation with 25(OH)D(3) versus vitamin D(3): effects on 25(OH)D levels, lower extremity function, blood pressure and markers of innate immunity. Journal of Bone and Mineral Research 2012 27 160-169. (doi:10.1002/jbmr.551)
• 14 Hewison M. An update on vitamin D and human immunity. Clinical Endocrinology 2012 76 315-325. (doi:10.1111/j.1365-2265.2011.04261.x)
• 15 Melamed ML, Michos ED, Post W & Astor B. 25-Hydroxyvitamin D levels and the risk of mortality in the general population. Archives of Internal Medicine 2008 168 1629-1637 (doi:10.1001/archinte.168.15.1629)
• 16 Dobnig H, Pilz S, Scharnagl H, Renner W, Seelhorst U, Wellnitz B, Kinkeldei J, Boehm BO, Weihrauch G & Maerz W. Independent association of low serum 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D levels with all-cause and cardiovascular mortality. Archives ofInternal Medicine 2008 168 1340-1349. (doi:10.1001/archinte.168.12.1340)
• 17 Hanwell HEC, Vieth R, Cole DE, Scillitani A, Modoni S, Frusciante V, Ritrovato G, Chiodini I, Minisola S & Carnevale V. Sun exposure questionnaire predicts circulating 25-hydroxy-vitamin D concentrations in Caucasian hospital workers in southern Italy. Journal ofSteroid Biochemistry and Molecular Biology 2010 121 334-337. (doi:10.1016/j.jsbmb.2010.03.023)
• 18 Carnevale V, Modoni S, Pileri M, Di Giorgio A, Chiodini I, Minisola S, Vieth R & Scillitani A. Longitudinal evaluation of vitamin D status in healthy subjects from southern Italy: seasonal and gender differences. Osteoporosis International 2001 12 1026-1030. (doi:10.1007/s001980170012)
• 19 Fraser WD & Milan AM. Vitamin D assays: past and present debates, difficulties, and developments. Calcified Tissue International 2013 92 118-127. (doi:10.1007/s00223-012-9693-3)
• 20 Lewis JG & Elder PA. Serum 25-OH vitamin D2 and D3 are stable under exaggerated conditions. Clinical Chemistry 2008 54 1931-1932. (doi:10.1373/clinchem.2008.111526)
• 21 Antoniucci DM, Black DM & Sellmeyer DE. Serum 25-hydroxy-vitamin D is unaffected by multiple freeze-thaw cycles. Clinical Chemistry 2005 51 258-261. (doi:10.1373/clinchem.2004. 041954)
• 22 Priemel M, von Domarus C, Klatte TO, Kessler S, Schlie J, Meier S, Proksch N, Pastor F, Netter C, Streichert T et al. Bone mineralization defects and vitamin D deficiency: histomorpho-metric analysis of iliac crest bone biopsies and circulating 25-hydroxyvitamin D in 675 patients. Journal of Bone andMineral Research 2010 25 305-312. (doi:10.1359/jbmr.090728)
• 23 Cavalier E, Wallace AM, Knox S, Mistretta VI, Cormier C &Souberbielle JC. Serum vitamin D measurement may not reflect what you give to your patients. Journal of Bone and Mineral Research 2008 23 1864-1865. (doi:10.1359/jbmr.080608)
• 24 Holick MF, Binkley NC, Bischoff-Ferrari HA, Gordon CM, Hanley DA, Heaney RP, Murad MH & Weaver CM. Guidelines for preventing and treating vitamin D deficiency and insufficiency revisited. Journal ofClinical Endocrinology and Metabolism 2012 97 1153-1158. (doi:10.1210/jc.2011-2601)
• 25 Hollis BW. Comparison of equilibrium and disequilibrium assay conditions for ergocalciferol, cholecalciferol and their major metabolites. Journal of Steroid Biochemistry 1984 21 81-86.(doi:10.1016/0022-4731(84)90063-3)
• 26 Kleerekoper M, Schleicher RL, Eisman J, Bouillon R, Singh RJ & Holick MF. Clinical applications for vitamin D assays: what is known and what is wished for. Clinical Chemistry 2011 57 1227-1232. (doi:10.1373/clinchem.2010.154997)
• 27 Romagnoli E, Mascia ML, Cipriani C, Fassino V, Mazzei F, D'Erasmo E, Carnevale V, Scillitani A & Minisola S. Short and long-term variations in serum calciotropic hormones after a single very large dose of ergocalciferol (vitamin D2) or cholecalciferol (D3) in the elderly. Journal of Clinical Endocrinology and Metabolism 2008 93 3015-3020. (doi:10.1210/jc.2008-0350)
• 28 Cipriani C, Romagnoli E, Scillitani A, Chiodini I, Clerico R, Carnevale V Mascia ML, Battista C, Viti R, Pileri M et al. Effect of a single oral dose of 600,000 IU of cholecalciferol on serum calciotropic hormones in young subjects with vitamin D deficiency: a prospective intervention study. Journal of Clinical Endocrinology and Metabolism 2010 95 4771-4777. (doi:10.1210/jc.2010-0502)
• 29 Petkovich MP & Jones G. CYP24A1 and chronic kidney disease. Current Opinion in Nephrology and Hypertension 2011 20 337-344. (doi:10.1097/MNH.0b013e3283477a7b)
• 30 Schlingmann KP, Kaufmann M, Weber S, Irwin A, Goos C, John U, Misselwitz J, Klaus G, Kuwertz-Broking E, Fehrenbach H et al. Mutations in CYP24A1 and idiopathic infantile hypercalce-mia. New England Journal of Medicine 2011 365 410-421. (doi:10.1056/NEJMoa1103864)
• 31 Tebben PJ, Milliner DS, Horst RL, Harris PC, Singh RJ, Wu Y, Foreman JW, Chelminski PR & Kumar R. Hypercalcemia, hypercalciuria, and elevated calcitriol concentrations with autosomal dominant transmission due to CYP24A1 mutations: effects of ketoconazole therapy. Journal of Clinical Endocrinology and Metabolism 2012 97 E423-E427. (doi:10.1210/jc.2011-1935)
• 32 Wang TJ, Zhang F, Richards JB, Kestenbaum B, van Meurs JB, Berry D, Kiel DP, Streeten EA, Ohlsson C, Koller DL et al. Common genetic determinants of vitamin D insufficiency: a genome-wide association study. Lancet 2010 376 180-188. (doi:10.1016/S0140-6736(10)60588-0)
• 33 Lensmeyer G, Poquette M, Wiebe D & Binkley N. The C-3 epimer of 25-hydroxyvitamin D3 is present in adult serum. Journal of Clinical Endocrinology and Metabolism 2012 97 163-168.(doi:10.1210/jc.2011-0584)
• 34 Carter GD. 25-Hydroxyvitamin D: a difficult analyte. Clinical Chemistry 2012 53 486-488. (doi:10.1373/clinchem.2011. 180562)
• 35 Farrell CJL, Martin S, McWhinney B, Straub I, Williams P & Herrmann M. State-of-the-art vitamin D assays: a comparison of www.eje-online.org automated immunoassays with liquid chromatography-tandem mass spectrometry methods. Clinical Chemistry 2012 58 531-542. (doi:10.1373/clinchem.2011.172155)
• 36 Binkley N, Krueger D, Cowgill CS, Plum L, Lake E, Hansen KE, DeLuca HF & Drezner MK. Assay variation confounds the diagnosis of hypovitaminosis D: a call for standardization. Journal of Clinical Endocrinology and Metabolism 2004 89 3152-3157. (doi:10.1210/jc.2003-031979)
• 37 Binkley N, Krueger DC, Morgan S & Wiebe D. Current status of clinical 25-hydroxyvitamin D measurement an assessment of between laboratory agreement. Clinica Chimica Acta 2010 4111976-1982. (doi:10.1016/j.cca.2010.08.018)
• 38 Van Schoor NM & Lips P. Worldwide vitamin D status. Best Practice & Research. Clinical Endocrinology & Metabolism 2011 25 671-680. (doi:10.1016/j.beem.2011.06.007)
• 39 Perna L, Haug U, Schottker B, Muller H, Raum E, Jansen EH & Brenner H. Public health implications ofstandardized 25-hydroxy-vitamin D levels: a decrease in the prevalence of vitamin D deficiency among older women in Germany. Preventive Medicine 2012 55 228-232. (doi:10.1016/j.ypmed.2012.06.010)
• 40 Phinney KW, Bedner M, Tai SS, Vamathevan VV, Sander LC, Sharpless KE, Wise SA, Yen JH, Schleicher RL, Chaudhary-Webb M et al. Development and certification of a standard reference material for vitamin D metabolites in human serum. Analytical Chemistry 2012 17 956-962. (doi:10.1021/ac202047n)
• 41 Sempos CT, Vesper HW, Phinney KW, Thienpont LM, Coates PM& Vitamin D Standardization Program (VDSP). Vitamin D status as an international issue: national surveys and the problem of standardization. Scandinavian Journal of Clinical and Laboratory Investigation 2012 72 (Suppl 243) 32-40.
• 42 Chun RF. New perspectives on the vitamin D binding protein. Cell Biochemistry andFunction 2012 30 445-456. (doi:10.1002/cbf. 2835)
• 43 White P & Cooke N. The multifunctional properties and characteristics of vitamin D-binding protein. Trends in Endocrinology and Metabolism 2000 11 320-327. (doi:10.1016/ S1043-2760(00)00317-9)
• 44 Chun RF, Peercy BE, Adams JS & Hewison M. Vitamin D binding protein and monocyte response to 25-hydroxyvitamin D and 1,25-dihydroxyvitaminD: analysisby mathematicalmodeling. PLoS ONE 2012 7 e30773. (doi:10.1371/journal.pone. 0030773)
• 45 Ahn J, Yu K, Stolzenberg-Solomon R, Simon KC, McCullough ML, Gallicchio L, Jacobs EJ, Ascherio A, Helzlsouer K, Jacobs KB et al. Genome-wide association study of circulating vitamin D levels. Human Molecular Genetics 2010 19 2739-2745. (doi:10.1093/ hmg/ddq155)
• 46 Powe CE, Ricciardi C, Berg AH, Erdenesanaa D, Collerone G, Ankers E, Wenger J, Karumanchi SH, Thadhani R & Bhan I. Vitamin D-binding protein modifies the vitamin D-bone mineral density relationship. Journal ofBone and Mineral Research 2011 26 1609-1616. (doi:10.1002/jbmr.387)
• 47 Levin GP, Robinson-Cohen C, De Boer IH, Houston DK, Lohman K, Liu Y, Kritchevsky SB, Cauley JA, Tanaka T, Ferrucci L et al. Genetic variants and associations of 25-hydroxy-vitamin D concentrations with major clinical outcomes. Journal of the American Medical Association 2012 308 1898-1905. (doi:10.1001/jama.2012.17304)
• 48 Wimalawansa SJ. Vitamin D in the new millennium. Current Osteoporosis Reports 2012 10 4-15. (doi:10.1007/s11914-011-0094-8)
• 49 Heaney RP. What is vitamin D insufficiency? And does it matter? Calcified Tissue International 2013 92 177-183. (doi:10.1007/ s00223-012-9605-6)
• 50 Rosen CJ & Taylor CL. Common misconceptions about vitamin D. Implications for clinicians. Nature Reviews. Endocrinology 2013 9 434-438.
• 51 Haines ST & Park SK. Vitamin D supplementation: what's known, what to do, and what's needed. Pharmacotherapy 2012 32 354-382. (doi:10.1002/phar.1037) Vitamin D assessment and supplementation R67
• 52 Souberbielle JC, Cormier C, Kindermans C, Gao P, Cantor T, Forette F & Baulieu EE. Vitamin D status and redefining serum parathyroid hormone reference range in the elderly. Journal of Clinical Endocrinology and Metabolism 2001 86 3086-3090. (doi:10.1210/jc.86.7.3086)
• 53 Valcour A, Blocki F, Hawkins DM & Rao SD. Effects of age and serum 25-OH-vitamin D on serum parathyroid hormone levels. Journal ofClinical Endocrinology and Metabolism 2012 97 3989-3995. (doi:10.1210/jc.2012-2276)
• 54 Brouwer-Brolsma EM, Bischoff-Ferrari HA, Bouillon R, Feskens EJM, Gallagher CJ, Hypponen E, Llewellyn DJ, Stoecklin E, Dierkes J, Kies AK et al. Vitamin D: do we get enough? A discussion between vitamin D experts in order to make a step towards the harmonisation of dietary reference intakes for vitamin D across Europe. Osteoporosis International 2013 24 1567-1577. (doi:10.1007/s00198-012-2231-3)
• 55 Adami S, Romagnoli E, Carnevale V, Scillitani A, Giusti A, Rossini M, Gatti D, Nuti R & Minisola S. Guidelines on prevention and treatment of vitamin D deficiency. Reumatismo 2011 63 129-147. (doi:10.4081/reumatismo.2011.129)
• 56 Rizzoli R, Boonen S, Brandi ML, Bruyere O, Cooper C, Kanis JA, Kaufman JM, Ringe JD, Weryha G & Reginster JY. Vitamin D supplementation in elderly or postmenopausal women: a 2013 update of the 2008 recommendations from the European Society for Clinical and Economic Aspects of Osteoporosis and Osteo-arthritis (ESCEO). Current Medical Research and Opinion 2013 29 305-313. (doi:10.1185/03007995.2013.766162)
• 57 Dawson-Hughes B, Mithal A, Bonjour JP, Boonen S, Burckhardt P, Fuleihan GEH, Josse RG, Lips P, Morales-Torres J & Yoshimura N. IOF position statement: vitamin D recommendations for older adults. Osteoporosis International 2010 21 1151-1154. (doi:10.1007/s00198-010-1285-3)
• 58 Ross AC, Taylor CL, Yaktine AL & Del Valle HB (Eds). Institute of Medicine. In Dietary Reference Intakes for Calcium and Vitamin D. Washington, DC: The National Academies Press, 2011.
• 59 Holick MF, Binkley NC, Bischoff-Ferrari HA, Gordon CM, Hanley DA, Heaney RP, Murad MH & Weaver CM. Evaluation,
• treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. Journal ofClinical Endocrinology and Metabolism 2011 96 1911-1930. (doi:10.1210/jc.2011-0385)
• 60 Pramyothin P & Holick MF. Vitamin D supplementation: guidelines and evidence for subclinical deficiency. Current Opinion in Gastroenterology 2012 28 139-150. (doi:10.1097/MOG. 0b013e32835004dc)
• 61 Pludowski P, HolickMF, Pilz S, Wagner CL, Hollis BW, Grant WB, Shoenfeld Y, Lerchbaum E, Llewellyn DJ, Kienreich K et al. Vitamin D effects on musculoskeletal health, immunity, auto-immunity, cardiovascular disease, cancer, fertility, pregnancy, dementia and mortality - a review of recent evidence. Autoimmunity Reviews 2013. In press. (doi:10.1016/j.autrev.2013.02.004)
• 62 Aloia JF. The 2011 report on dietary reference intake for vitamin D: where do we go from here? Journal of Clinical Endocrinology and Metabolism 2011 96 2987-2996. (doi:10.1210/jc.2011-0090)
• 63 Rosen CJ, Abrams SA, Aloia JF, Brannon PM, Clinton SK, *Durazo-Arvizu RA, Gallagher JC, Gallo RL, Jones G, Kovacs CS et al. IOM committee members respond to Endocrine Society vitamin D guideline. Journal of Clinical Endocrinology and Metabolism 2012 97 1146-1152. (doi:10.1210/jc.2011-2218)
• 64 Ross AC, Manson JE, Abrams SA, Aloia JF, Brannon PM, Clinton SK, Durazo-Arvizu RA, Gallagher JC, Gallo RL, Jones G et al. The 2011 report on dietary reference intakes for calcium and vitamin D from the Institute of Medicine: what clinicians need to know. Journal of Clinical Endocrinology and Metabolism 2011 96 53-58. (doi:10.1210/jc.2010-2704)
• 65 Vieth R. Why the minimum desirable serum 25-hydroxyvitamin D level should be 75 nmol/L (30 ng/ml). Best Practice & Research. Clinical Endocrinology & Metabolism 2011 25 681-691. (doi:10.1016/j.beem.2011.06.009) www.eje-online.org R68 E Romagnoli and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2013) 169
• 66 Bouillon R. Why modest but widespread improvement of the vitamin D status is the best strategy? Best Practice & Research. Clinical Endocrinology & Metabolism 2011 25 693-702. (doi:10.1016/j.beem.2011.06.008)
• 67 Vanlint S. Vitamin D and obesity. Nutrients 2013 5 949-956. (doi:10.3390/nu5030949)
• 68 Heaney RP. Vitamin D - baseline status and effective dose. New England Journal of Medicine 2012 367 77-78. (doi:10.1056/ NEJMe1206858)
• 69 Autier P, Gandini S & Mullie P. A systematic review: influence of vitamin D supplementation on serum 25-hydroxyvitamin D concentration. Journal of Clinical Endocrinology and Metabolism 2012 97 2606-2613. (doi:10.1210/jc.2012-1238)
• 70 Rosen CJ. Vitamin D insufficiency. New England Journal of Medicine 2011 364 248-254. (doi:10.1056/NEJMcp1009570)
• 71 Souberbielle JC & Cavalier E. Supplementation, optimal status, and analytical determination of vitamin D: where are we standing in 2012? Anti-Cancer Agents in Medicinal Chemistry 2013 13 36-44. (doi:10.2174/187152013804487317)
• 72 Sinha A, Cheetham TD & Pearce SHS. Prevention and treatment of vitamin D deficiency. Calcified Tissue International 2013 92 207-215. (doi:10.1007/s00223-012-9663-9)
• 73 Hackman KL, Gagnon C, Briscoe RK, Lam S, Anpalahan M & Ebeling PR. Efficacy and safety of oral continuous low-dose versus short-term high-dose vitamin D: a prospective randomised trial conducted in a clinical setting. Medical Journal of Australia 2010 192 686-689.
• 74 Pekkarinen T, Valimaki VV, Aarum S, Turpeinen U, Hamalainen E, Loyttyniemi E & Valimaki MJ. The same annual dose of 292000 IU of vitamin D (cholecalciferol) on either daily or four monthly basis for elderly women: 1-year comparative study of the effects on serum 25(OH)D concentrations and renal function. Clinical Endocrinology 2010 72 455-461.(doi:10.1111/j.1365-2265.2009.03637.x)
• 75 Ilahi M, Armas LA & Heaney RP. Pharmacokinetics of a single, large dose of cholecalciferol. American Journal of Clinical Nutrition 2008 87 688-691.
• 76 Sanders KM, Stuart AL, Williamson EJ, Simpson JA, Kotowicz MA, Young D & Nicholson GC. Annual high-dose oral vitamin D and falls and fractures in older women: a randomized controlled trial. Journal ofthe American Medical Association 2010 303 1815-1822. (doi:10.1001/jama.2010.594)
• 77 Smith H, Anderson F, Raphael H, Maslin P, Crozier S & Cooper C. Effect of annual intramuscular vitamin D on fracture risk in elderly men and women: a population-based, randomized, double-blind, placebo-controlled trial. Rheumatology 2007 46 1852-1857. (doi:10.1093/rheumatology/kem240)
• 78 Sanders KM, Nicholson GC & Ebeling PR. Is high dose vitamin D harmful? Calcified Tissue International 2013 92 191-206. (doi:10.1007/s00223-012-9679-1)
• 79 Hansen KE. High-dose vitamin D: helpful or harmful? Current Rheumatology Reports 2011 13 257-264. (doi:10.1007/ s11926-011-0175-9)
• 80 Dawson-Hughes B & Harris SS. High-dose vitamin D supplementation: too much of a good thing? Journal of the American Medical Association 2010 303 1861-1862. (doi:10.1001/jama. 2010.598)
• 81 Minisola S, Colangelo L, Cilli M, Cipriani C, Pepe J & Romagnoli E. Intermittent high doses of vitamin D: a need for further studies? Calcified Tissue International 2013 92 487-488. (doi:10.1007/s00223-013-9714-x)
• 82 Heaney RP & Holick MF. Why the IOM recommendations for vitamin D are deficient. Journal of Bone and Mineral Research 2011 26 455-457. (doi:10.1002/jbmr.328)
• 83 Holick MF. The D-batable Institute of Medicine Report: a D-lightful perspective. Endocrine Practice 2011 17 143-149. (doi:10.4158/EP.17.1.143)
• 84 Lagari VS, GiSmez-Marin O & Levis S. Differences in vitamin D3 dosing regimens in a geriatric community-dwelling population. Endocrine Practice 2012 18 847-854. (doi:10.4158/EP12081.OR)
• 85 Binkley N, Gemar D, Engelke J, Gangnon R, Ramamurthy R, Krueger D & Drezner MK. Evaluation of ergocalciferol or cholecalciferol dosing 1,600 IU daily or 50,000 IU monthly in older adults. Journal o f ClinicalEndocrinology and Metabolism 2011 96 981-988. (doi:10.1210/jc.2010-0015)
• 86 Cavalier E, Fache W & Souberbielle JC. A randomised, double-blinded, placebo-controlled, parallel study of vitamin D3 supplementation with different schemes based on multiples of 25,000 IU doses. International Journal of Endocrinology 2013. In press. (doi:10.1155/2013/327265)
• 87 Rolland Y, de Souto Barreto P, Abellan Van Kan G, Annweiler C, Beauchet O, Bischoff-Ferrari H, Berrut G, Blain H, Bonnefoy M, Cesari M et al. Vitamin D supplementation in older adults: searching for specific guidelines in nursing homes. Journal of Nutrition, Health & Aging 2013 17 402-412. (doi:10.1007/s12603-013-0007-x)
• 88 Houghton LA & Vieth R. The case against ergocalciferol (vitamin D2) as a vitamin supplement. American Journal of Clinical Nutrition 2006 84 694-697.
• 89 Tripkovic L, Lambert H, Hart K, Smith CP, Bucca G, Penson S, Chope G, Hypponen E, Berry J, Vieth R et al. Comparison of vitamin D2 and vitamin D3 supplementation in raising serum 25-hydroxyvitamin D status: a systematic review and meta-analysis. American Journal of Clinical Nutrition 2012 95 1357-1364. (doi:10.3945/ajcn.111.031070)
• 90 Holmberg I, Berlin T, Ewerth S & Bjorkhem I. 25-Hydroxylase activity in subcellular fractions from human liver. Evidence for different rates of mitochondrial hydroxylation of vitamin D2 and D3. Scandinavian Journal of Clinical and Laboratory Investigation 1986 46 785-790. (doi:10.3109/00365518 609084051)
• 91 Horst RL, Reinhardt TA, Ramberg CF, Koszewski NJ & Napoli JL. 24-Hydroxylation of 1,25 dihydroxyergocalciferol - an unambiguous deactivation process. Journal ofBiological Chemistry 1986 261 9250-9256.
• 92 Russo S, Carlucci L, Cipriani C, Ragno A, Piemonte S, Del Fiacco R, Pepe J, Fassino V, Arima S, Romagnoli E et al. Metabolic changes following 500 mg monthly administration of calcidiol: a study in normal females. Calcified Tissue International 2011 89 252-257. (doi:10.1007/s00223-011-9513-1)
• 93 Diamond TH, Ho KW, Rohl PG & Meerkin M. Annual intramuscular injection of a megadose of cholecalciferol for treatment of vitamin D deficiency: efficacy and safety data. Medical Journal of Australia 2005 183 10-12.
• 94 de Paula FJ & Rosen CJ. Vitamin D safety and requirements. Archives of Biochemistry and Biophysics 2012 523 64-72. (doi:10.1016/j.abb.2011.12.002)
• 95 Zittermann A, Prokop S, Gummert JF & Borgermann J. Safety issues of vitamin D supplementation. Anti-Cancer Agents in *Medicinal Chemistry 2013 13 4-10. (doi:10.2174/1871520 13804487290)
• 96 Heaney RP, Davies KM, Chen TC, Holick MF & Barger-Lux MJ. Human serum 25-hydroxycholecalciferol response to extended oral dosing with cholecalciferol. American Journal of Clinical Nutrition 2003 77 204-210.
• 97 Cashman KD & Kiely M. Towards prevention of vitamin D deficiency and beyond: knowledge gaps and research needs in vitamin D nutrition and public health. British Journal ofNutrition 2011 106 1617-1627. (doi:10.1017/S0007114511004995)
• 98 Rollins G. Vitamin D testing: what's the right answer? Labs grapple with confusing analytics, evidence. Clinical Laboratory News 2009 35 1, 6, 8.
• 99 Maxmen A. The vitamin D-lemma. Nature 2011 475 23-25. (doi:10.1038/475023a)
• 100 Corbet-Dooren J. Supplements may not prevent bone fractures. Wall Street Journal 2013 (2013-02-26).
• 101 Findlay M, Anderson J, Roberts S, Almond A & Isles C. Treatment of vitamin D deficiency: divergence between clinical practice and expert advice. Postgraduate Medical Journal 2012 88 255-260. (doi:10.1136/postgradmedj-2011-130243) www.eje-online.org EUROPEAN JOURNAL OF ENDOCRINOLOGY (2013) 169
• 102 Gallagher JC, Yalamanchili V & Smith LM. The effect of vitamin D on calcium absorption in older women. Journal of Clinical Endocrinology and Metabolism 2012 97 3550-3556. (doi:10.1210/jc.2012-2020)
• 103 Need AG, O'Loughlin PD, Morris HA, Coates PS, Horowitz M & Nordin BE. Vitamin D metabolites and calcium absorption in severe vitamin D deficiency. Journal of Bone and Mineral Research 2008 23 1859-1863. (doi:10.1359/jbmr.080607)
• 104 Heaney RP, Dowell MS, Hale CA & Bendich A. Calcium absorption varies within the reference range for serum 25-hydroxyvitamin D. Journal ofthe American College ofNutrition 2003 22 142-146. (doi:10.1080/07315724.2003.10719287)
• 105 Sai AJ, Walters RW, Fang X & Gallagher JC. Relationship between vitamin D, parathyroid hormone, and bone health. Journal of Clinical Endocrinology and Metabolism 2011 96 E436-E446. (doi:10.1210/jc.2010-1886)
• 106 Carnevale V, Nieddu L, Romagnoli E, Battista C, Mascia ML, Chiodini I, Eller-Vainicher C, Frusciante V, Santini SA, La Porta M et al. Regulation of PTH secretion by 25-hydroxyvitamin D and ionized calcium depends on vitamin D status: a study in a large cohort of healthy subjects. Bone 2010 47 626-630. (doi:10.1016/j.bone.2010.06.013)
• 107 Dawson-Hughes B, Heaney RP, Holick MF, Lips P, Meunier PJ & Vieth R. Estimates of optimal vitamin D status. Osteoporosis International2005 16 713-716. (doi:10.1007/s00198-005-1867-7)
• 108 DIPART (Vitamin D Individual Patient Analysis of Randomized Trials) Group. Patient level pooled analysis of 68 500 patients from seven major vitamin D fracture trials in US and Europe. BMJ 2010 340 b5463. (doi:10.1136/bmj.b5463)
• 109 Prentice RL, Pettinger MB, Jackson RD, Wactawski-Wende J, LaCroix AZ, Anderson GL, Chlebowski RT, Manson JE, Van Horn L, Vitolins MZ et al. Health risks and benefits from calcium and vitamin D supplementation: Women's Health Initiative clinical trial and cohort study. Osteoporosis International 2013 24 567-580. (doi:10.1007/s00198-012-2224-2)
• 110 Moyer VA & on behalf of the US Preventive Services Task Force. Vitamin D and calcium supplementation to prevent fractures in adults: U.S. Preventive Services Task Force Recommendation Statement. Annals of Internal Medicine 2013. In press. (doi:10.7326/0003-4819-159-6-201309170-00685)
• 111 Dawson-Hughes B. What is the optimal dietary intake of vitamin D for reducing fracture risk? Calcified Tissue International 2013 92 184-190. (doi:10.1007/s00223-012-9606-5)
• 112 Bischoff-Ferrari HA, Willett WC, Orav EJ, Lips P, Meunier PJ, Lyons RA, Flicker L, Wark J, Jackson RD, Cauley JA et al. A pooled analysis of vitamin D dose requirements for fracture prevention. New England Journal of Medicine 2012 367 40-49. (doi:10.1056/NEJMoa1109617) Vititamin D assessment and supplementation R69
• 113 Gallagher JC. Vitamin D deficiency and muscle strength: are they related? Journal ofClinical Endocrinology and Metabolism 2012 97 4366-4369. (doi:10.1210/jc.2012-3720)
• 114 Murad MH, Elamin KB, Abu Elnour NO, Elamin MB, Alkatib AA, Fatourechi MM, Almandoz JP, Mullan RJ, Lane MA, Liu H et al. The effect of vitamin D on falls: a systematic review and meta-analysis. Journal of Clinical Endocrinology and Metabolism 2011 96 2997-3006. (doi:10.1210/jc.2011-1193)
• 115 Bischoff-Ferrari HA, Dawson-Hughes B, Staehelin HB, Orav JE, Stuck AE, Theiler R, Wong JB, Egli A, Kiel DP & Henschkowski J. Fall prevention with supplemental and active forms of vitamin D: a meta-analysis of randomised controlled trials. BMJ2009 339 b3692. (doi:10.1136/bmj.b3692)
• 116 Cooper L, Clifton-Bligh PB, Nery ML, Figtree G, Twigg S, Hibbert E & Robinson BG. Vitamin D supplementation and bone mineral density in early postmenopausal women. American Journal ofClinical Nutrition 2003 77 1324-1329.
• 117 Ooms ME, Roos JC, Bezemer PD, van der Vijgh WJ, Bouter LM & Lips P. Prevention of bone loss by vitamin D supplementation in elderly women: a randomized double-blind trial. Journal of Clinical Endocrinology and Metabolism 1995 80 1052-1058.(doi:10.1210/jc.80.4.1052)
• 118 Peacock M, Liu G, Carey M, McClintock R, Ambrosius W, Hui S & Johnston CC. Effect of calcium or 25OH vitamin D3 dietary supplementation on bone loss at the hip in men and women over the age of 60. Journal ofClinical Endocrinology and Metabolism 2000 85 3011-3019. (doi:10.1210/jc.85.9.3011)
• 119 Bischoff-Ferrari HA, Kiel DP, Dawson-Hughes B, Orav JE, Li R, Spiegelman D, Dietrich T & Willett WC. Dietary calcium and serum 25-hydroxyvitamin D status in relation to BMD among U.S. adults. Journal of Bone and Mineral Research 2009 24 935-942. (doi:10.1359/jbmr.081242)
• 120 Joo NS, Dawson-Hughes B, Kim YS, Oh K & Yeum KJ. Impact of calcium and vitamin D insufficiencies on serum parathyroid hormone and bone mineral density: analysis of the fourth and fifth Korea National Health and Nutrition Examination Survey (KNHANES IV-3, 2009 and KNHANES V-1, 2010). Journal of Bone and Mineral Research 2013 28 764-770. (doi:10.1002/ jbmr.1790)