Vitamin D metabolism

Vitamin D is a fat-soluble, seco-steroid hormone. The term vitamin D refers to two precursors, i.e. cholecalciferol and ergocalciferol. Cholecalciferol is mostly formed in the skin after exposure to sunlight. In the skin, the precursor 7-dehydrocholesterol is transformed into cholecalciferol under the influence of short-wave UV light⁽Reference Lips¹⁾. Another source of vitamin D is the diet. Ergocalciferol is generated in yeast and plants and cholecalciferol is produced in fish and mammals. In general, oral vitamin D intake, especially in Europe, is low and depends mostly on cutaneous production of vitamin D for our reserves⁽Reference Lips²⁾. The inert precursors are transported to the liver, where they are converted to 25-hydroxyvitamin D₃ (25OHD₃). In the kidney, 25OHD₃ is hydroxylated by the enzyme 25OHD₃-1α-hydroxylase (1α-OHase) to form 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), the most active vitamin D metabolite (Fig. 1). 1α-OHase expression is not restricted to the kidney. Several cell types like macrophages, osteoblasts and neurons have also been shown to express 1α-OHase (Table 1) ⁽Reference Hewison^3–Reference McCann and Ames⁵⁾.

Fig. 1 Different pathways for the activation of vitamin D. Inert vitamin D precursors are either formed in the skin after exposure to UVB or derived form the diet. These precursors are hydroxylated in the liver to form 25-hydroxyvitamin D₃ (25(OH)D₃). The 25(OH)D₃ is bound to the vitamin D-binding protein (DBP). The final 1-α-hydroxylation step that forms the most active vitamin D metabolite, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) occurs either in the kidney (bulk) or in the extra-renal cells expressing the 1-α-hydroxylase enzyme. The 1,25(OH)₂D₃ formation in the kidney is tightly regulated via a feedback mechanism and the active vitamin D formed in the kidney exerts endocrine effects after binding to the vitamin D receptor (VDR). The VDR forms a heterodimer with the retinoid receptor (RXR) and regulates gene transcription. The active vitamin D formed extra-renally exerts paracrine and autocrine effects. IGF, insulin-like growth factor; FGF, fibroblast growth factor; PTH, parathyroid hormone; 24,25(OH)₂D₃, 24,25-dihydroxyvitamin D₃.

Table 1 Extra-renal expression of the 1α-hydroxylase (1α-OHase) enzyme and effects of potential regulators relevant for ageing*

*Based on ex vivo, in vitro and animal studies.

1,25(OH)₂D₃, 1,25-dihydroxyvitamin D₃; TGFβ1, transforming growth factor β1; IFN-γ, interferon-γ; PTH, parathyroid hormone; EGF, epidermal growth factor; ↑ , stimulating effect; ↓ , inhibiting effect; –, no effect; (?), uncertain.

The primary function of the vitamin D endocrine system is maintaining Ca and phosphate homeostasis. Vitamin D stimulates both intestinal absorption and renal reabsorption of Ca and phosphate. Vitamin D deficiency results in decreased Ca and phosphate (re)absorption and subsequently lower serum levels of Ca and phosphate. This stimulates parathyroid hormone (PTH) secretion from the parathyroid glands⁽Reference Holick⁶⁾. PTH stimulates renal 1α-OHase expression and 1,25(OH)₂D₃ formation. PTH also stimulates osteoclast formation (osteoclastogenesis). Osteoclasts stimulate bone resorption, releasing Ca and phosphate ions from the bone into the blood. A recent animal study has demonstrated that osteoclastogenesis was increased in mice when serum 25OHD₃ levels were < 80 nmol/l and this was positively associated with the receptor activator for NF-κB ligand/osteoprotegerin ratio. This increase in bone resorption was associated with the development of osteopenia and osteoporosis⁽Reference Anderson, Sawyer and Moore⁷⁾. The optimal serum 25OHD₃ level in human subjects to prevent stimulation of osteoclastogenesis is also believed to be about 80 nmol/l⁽Reference Anderson, Sawyer and Moore⁷⁾.

Other hormones that are known to stimulate renal 1α-OHase expression are insulin-like growth factor 1, calcitonin and oestrogen⁽Reference Gomez^8, Reference Gallagher, Riggs and DeLuca⁹⁾. Increases in serum Ca, phosphate and 1,25(OH)₂D₃ levels down-regulate renal 1α-OHase expression. Serum 1,25(OH)₂D₃ levels are also regulated by the enzyme 25OHD₃-24-hydroxylase (24OHase). Expression of 24OHase in the kidney is stimulated by 1,25(OH)₂D₃ and this enzyme converts 1,25(OH)₂D₃ into less active metabolites. These feedback mechanisms play an important role in the protection against hypercalcaemia and hyperphosphataemia⁽Reference Christakos, Dhawan and Benn¹⁰⁾.

Extra-renal 1α-OHase expression and activity is modulated differently from renal 1α-OHase and is less sensitive to feedback regulation by 1,25(OH)₂D₃⁽Reference Hewison, Zehnder and Bland¹¹⁾. It is suggested that induction of extra-renal 1α-OHase involves regulatory pathways that differ from the renal, cyclic AMP-mediated pathway. For example, in osteoblasts, 1α-OHase expression and activity is not influenced by the levels of 1,25(OH)₂D₃, PTH and Ca like renal 1α-OHase. IL1β, an activator of NF-κB, stimulates both 1α-OHase expression and activity in osteoblasts⁽Reference van Driel, Koedam and Buurman⁴⁾. In macrophages, immune signals such as TNFα and interferon-γ modulate 1α-OHase expression, while in vascular smooth muscle cells extra-renal 1α-OHase expression and activity is stimulated by the hormones PTH and oestrogen (Table 1)⁽Reference Somjen, Weisman and Kohen^12, Reference Zehnder, Bland and Williams¹³⁾. Regulators of 1α-OHase expression and activity in most extra-renal tissues and the functions of the extra-renally formed 1,25(OH)₂D₃ are still largely unknown. Some age-related effects on extra-renal 1α-OHase expression have been reported⁽Reference Somjen, Katzburg and Stern¹⁴⁾. In an animal model, ageing resulted in decreased bone 1α-OHase expression⁽Reference Anderson, O'Loughlin and May¹⁵⁾. Specific effects of ageing on the 1α-OHase expression and the activity in extra-renal tissues and health consequences of alterations in 1α-OHase expression and activity with ageing remain to be elucidated.

In recent years, several proteins have been discovered, which are important regulators of both Ca and phosphate homeostasis and the vitamin D endocrine system. These are fibroblast growth factor-23 (FGF-23) and klotho, a β-glucuronidase⁽Reference Chang, Hoefs and van der Kemp^16, Reference Yamashita, Yoshioka and Itoh¹⁷⁾. FGF-23 is involved in the regulation of renal phosphate excretion. FGF-23 inhibits expression of the renal sodium-phosphate transporter and thereby increases phosphate excretion⁽Reference Lanske and Razzaque¹⁸⁾. In addition, FGF-23 decreases renal 1α-OHase expression, stimulates 24OHase expression and decreases both PTH mRNA expression and PTH secretion from the parathyroid gland⁽Reference Lanske and Razzaque^18–Reference Shimada, Hasegawa and Yamazaki²⁰⁾. This leads to lower 1,25(OH)₂D₃ levels and thus decreases vitamin D-related effects on Ca and phosphate homeostasis. Klotho is involved in the regulation of renal Ca absorption and acts as a co-receptor or cofactor for other proteins such as FGF-23⁽Reference Urakawa, Yamazaki and Shimada^21, Reference Nabeshima and Imura²²⁾. Klotho is capable of binding to various FGF receptors and enhances FGF-23 signalling. FGF-23 and klotho thus have important functions in regulating Ca and phosphate homeostasis and are important for skeletal health, both via effects on the vitamin D endocrine system and via direct, non-vitamin D-dependent effects.

Effects of ageing on vitamin D metabolism

Vitamin D deficiency is a worldwide problem⁽Reference Holick⁶⁾. Although in Europe and the United States there has been strong attention on vitamin D in recent years and vitamin D-fortified food products are widely available, vitamin D deficiency is still very prevalent among older people⁽Reference Lanske and Razzaque^23–Reference Yetley²⁵⁾. Mean serum 25OHD₃ concentrations in The Netherlands in independent community-dwelling older people are about 30 nmol/l and in institutionalised older people about 20–25 nmol/l⁽Reference Chel, Wijnhoven and Smit^26, Reference Lips²⁷⁾. Similar serum 25OHD₃ levels among community-dwelling elderly have been reported in the United Kingdom and Germany⁽Reference Gallacher, McQuillian and Harkness^28, Reference Hintzpeter, Mensink and Thierfelder²⁹⁾. Reports suggest that serum 25OHD₃ levels in older people in the United States are higher than in Europe⁽Reference Chel, Wijnhoven and Smit^26, Reference Holick, Siris and Binkley^30, Reference Kuriacose and Olive³¹⁾. This is most likely due to higher oral intake of vitamin D in the United States where vitamin D fortification of food is more prevalent than that in Europe⁽Reference Holden, Lemar and Exler^24, Reference Calvo, Whiting and Barton³²⁾. However, even in the United States, >50 % of the community-dwelling elderly are reported to have serum 25OHD₃ levels < 75 nmol/l and about 30 % of the elderly have levels < 50 nmol/l⁽Reference Holick, Siris and Binkley³⁰⁾.

The high prevalence of vitamin D deficiency in older people may have several causes. Cholecalciferol synthesis in the skin after sun exposure is less effective in old age because of a decline in cutaneous levels of 7-dehydrocholesterol⁽Reference MacLaughlin and Holick³³⁾. The level in a 70-year-old is only approximately 25 % of the 7-dehydrocholesterol level in young persons⁽Reference Holick and Chen³⁴⁾. This is worsened by the decreased exposure to sunlight with ageing due to immobility, lack of transport and social isolation⁽Reference Holick, Matsuoka and Wortsman^35, Reference Adami, Giannini and Bianchi³⁶⁾. Another factor contributing to the increased risk of vitamin D deficiency is an increase in body fat with ageing. The increase in fat mass leads to a larger distribution volume for the fat-soluble 25OHD₃, which decreases the bioavailability of 25OHD₃. Consequently, an inverse association has been demonstrated between BMI and both serum levels 25OHD₃ and 1,25(OH)₂D₃ and a positive association between BMI and PTH levels has been demonstrated⁽Reference Konradsen, Ag and Lindberg^37, Reference Snijder, van Dam and Visser³⁸⁾.

An age-related decrease in 1,25(OH)₂D₃ levels has also been suggested but reports are conflicting⁽Reference Lau and Baylink³⁹⁾. When vitamin D levels are low, compensatory hyperparathyroidism increases renal conversion of 25OHD₃ to 1,25(OH)₂D₃ and thereby maintains normal or even slightly elevated levels of this metabolite. As vitamin D deficiency worsens, 1,25(OH)₂D₃ formation is impaired due to a lack of substrate⁽Reference Lau and Baylink³⁹⁾. Additionally, several age-related effects have been reported that could lead to lower 1,25(OH)₂D₃ levels with ageing. First, renal function declines with age and this is accompanied with a decline in renal 1α-OHase activity and thus impaired conversion of 25OHD₃ to 1,25(OH)₂D₃⁽Reference Dukas, Schacht and Mazor⁴⁰⁾. Second, levels of insulin-like growth factor 1, calcitonin and oestrogen, which stimulate 1α-OHase expression and activity, decrease with ageing⁽Reference Thorner and Nass⁴¹⁾. Furthermore, 1,25(OH)₂D₃ metabolism may increase with ageing. In an animal model, an age-dependent increase in renal 24OHase expression was reported. This occurred predominantly in female animals, suggesting an effect of ovarian hormones⁽Reference Matkovits and Christakos⁴²⁾. Ovariectomy in these animals was indeed associated with up-regulation of 24OHase expression. Interestingly, the induction of 24OHase by 1,25(OH)₂D₃ may also be affected by ageing⁽Reference Armbrecht and Boltz⁴³⁾.

Effects of ageing on vitamin D action

The active metabolite 1,25(OH)₂D₃ exerts its function via the vitamin D receptor (VDR), a nuclear receptor. Upon binding of 1,25(OH)₂D₃, the VDR forms a heterodimer with the retinoid receptor and binds to a vitamin D-responsive element in the promoter region of a target gene. This influences transcription of vitamin D-responsive genes⁽Reference Lips¹⁾. In addition, the functions of the VDR are not limited to the binding to vitamin D-responsive element. The VDR has also been found to bind β-catenin, a key transcriptional factor in the Wnt signalling pathway⁽Reference Bikle^44, Reference Shah, Islam and Dakshanamurthy⁴⁵⁾. This pathway has been implicated in a number of malignancies. By binding to β-catenin, the VDR blocks its transcriptional activity and so exerts antiproliferative properties. Besides genomic effects via the VDR, 1,25(OH)₂D₃ also exerts non-genomic effects via a membrane-bound plasma receptor or second messengers such as cyclic AMP. These are rapid effects that do not depend on gene transcription.

Almost all tissues and cells in the body express the VDR, including those not directly involved in the regulation of Ca homeostasis, enabling a broad range of effects. Extra-renal 1,25(OH)₂D₃ formation in various tissues implies that 1,25(OH)₂D₃ is also capable of exerting paracrine and autocrine effects in addition to the well-known endocrine effects. Among the paracrine and autocrine effects are regulation of cell proliferation, differentiation and apoptosis⁽Reference van Driel, Koedam and Buurman⁴⁶⁾.

Alterations in VDR expression leading to vitamin D resistance with ageing have received particular interest. With ageing, a decrease in VDR expression in bone, intestine and muscle tissue has been reported⁽Reference Duque, El Abdaimi and Macoritto^47–Reference Bischoff-Ferrari, Borchers and Gudat⁴⁹⁾. Various factors are known to influence VDR expression. Oestrogen, growth hormones and 1,25(OH)₂D₃ are stimulators of VDR expression but serum levels decrease with ageing⁽Reference Welsh, Wietzke and Zinser^50, Reference Klaus, Weber and Rodriguez⁵¹⁾. On the other hand, TNFα has been shown to down-regulate VDR expression, while serum TNFα levels increase with ageing⁽Reference Andress^52, Reference Fernandez-Martin, Kurian and Farmer⁵³⁾.

In addition to a decrease in VDR numbers, binding of 1,25(OH)₂D₃ to the VDR might also be decreased with ageing. A recent animal study, using competition VDR-binding assays with [³H]-1,25(OH)₂D₃, has reported a decrease in 1,25(OH)₂D₃ binding to the VDR with ageing in duodenal tissue⁽Reference Gonzalez Pardo, Boland and de Boland⁵⁴⁾. Whether this also occurs in human subjects is not known.

Parathyroid hormone and ageing

Like 25OHD₃, PTH levels also exhibit seasonal variation with the highest PTH levels observed during the winter months⁽Reference Vecino-Vecino, Gratton and Kremer⁵⁵⁾. A (secondary) rise in PTH levels is generally observed with ageing with a prevalence varying from 20 to 60 %⁽Reference Bjorkman, Sorva and Tilvis⁵⁶⁾. The most important causes of this secondary rise with ageing are vitamin D deficiency and resistance, renal insufficiency and low dietary intake of Ca⁽Reference Perez, Picotto and Carpentieri⁵⁷⁾. PTH stimulates 1,25(OH)₂D₃ formation and mobilises Ca from bone in order to maintain normal serum Ca levels⁽Reference Lips¹⁾. Hyperparathyroidism not only negatively influences bone health but also is associated with sarcopaenia and falls as PTH stimulates muscle protein breakdown⁽Reference Visser, Deeg and Lips⁵⁸⁾. Furthermore, hyperparathyroidism has been related to cardiovascular events as PTH has been shown to promote vascular calcification⁽Reference Rashid, Bernheim and Green⁵⁹⁾. Recently, elevated PTH levels have been shown to be an independent predictor of impaired long-term survival prognosis in older people⁽Reference Bjorkman, Sorva and Tilvis⁵⁶⁾. High serum PTH levels ( ≥ 63 ng/l) were associated with significant increases in mortality (hazard ratio = 1·56, 95 % CI: 1·29, 1·88) and a 2·3-year reduction of median life expectancy in a cohort of older patients⁽Reference Bjorkman, Sorva and Tilvis⁵⁶⁾.

Calcium homeostasis and ageing

Ca homeostasis involves a coordinated control of Ca handling by the intestine, kidney and bone under the influence of primarily PTH and 1,25(OH)₂D₃. Ageing, vitamin D deficiency and vitamin D resistance all affect these processes negatively. The two main mechanisms for Ca (re)absorption are a transcellular (active) and a paracellular (passive) route. The transcellular route involves entry of Ca into the cell at the apical side of the cell via Ca channels, diffusion of Ca through the cytosol bound to calbindins and active extrusion of Ca across the basolateral membrane via a Ca pump or a Na/Ca exchanger⁽Reference Perez, Picotto and Carpentieri⁵⁷⁾. The epithelial Ca channels are members of the transient receptor potential (TRP) super family and more precisely, the vanilloid subfamily (TRPV). The TRPV5 channel is the major isoform in the kidney, while the TRPV6 channel is highly expressed in the intestine. The paracellular route involves diffusion of Ca via tight junctions between epithelial cells.

What is vitamin D deficiency?

Measurement of serum 25OHD₃ level is the best clinical indicator to assess vitamin D status. Serum 25OHD₃ levels represent the combined contribution of both cutaneous synthesis and oral intake of the various dietary sources of vitamin D⁽Reference Zerwekh⁷⁹⁾. Levels of 1,25(OH)₂D₃ are less suitable to assess vitamin D status because even in a state of vitamin D deficiency 1,25(OH)₂D₃ levels can be normal or slightly elevated.

With the ever-increasing insights into the effects of vitamin D, optimal vitamin D status is becoming more difficult to define. Criteria and cut-off values for vitamin D deficiency have mostly been linked to the effects of PTH levels on bone turnover. Serum 25OHD₃ levels are inversely associated with PTH levels until an inflection point is reached. At this point, PTH levels begin to level off. Estimates for the serum 25OHD₃ concentration at which the PTH concentration becomes constant vary from 25 to 122 nmol/l⁽Reference Bjorkman, Sorva and Tilvis^80, Reference Adami, Viapiana and Gatti⁸¹⁾. This wide variation in estimates is due to inter-individual variation in Ca (re)absorption and vitamin D responsiveness, as previously discussed. Old people generally require higher serum 25OHD₃ levels, and thus vitamin D intake, to suppress PTH levels when compared with younger individuals. The capacity of the different vitamin D metabolites to raise serum 25OHD₃ levels is unaltered with ageing⁽Reference Vieth, Ladak and Walfish⁸²⁾.

When the effects on PTH levels and bone turnover are evaluated, serum 25OHD₃ concentrations of >50 nmol/l are regarded by many as sufficient⁽Reference Lips⁸³⁾. However, when other health benefits of vitamin D are taken into account, including its non-calcaemic effects, serum 25OHD₃ concentrations of >75 nmol/l are advised⁽Reference Heaney⁸⁴⁾. In addition, the process of extra-renal 1,25(OH)₂D₃ formation and autocrine and paracrine effects are most efficient when serum 25OHD₃ levels are >75 nmol/l⁽Reference Holick⁶⁾.

In many trials that study the effect of vitamin D supplementation, Ca intake is not measured, which complicates the comparison of individual trial results. Dietary Ca content has been shown to modulate the 25OHD₃/PTH association⁽Reference Adami, Viapiana and Gatti⁸¹⁾. As Ca intake is lower, higher 25OHD₃ serum levels are required to normalise PTH concentrations. In part, this may also explain discordant results between intervention trials with vitamin D, as Ca intake differs among countries⁽Reference Boonen, Lips and Bouillon^85, Reference Branca and Valtuena⁸⁶⁾.

Consequences of vitamin D deficiency and resistance

Vitamin D deficiency and resistance have important consequences for older people (Fig. 2). To illustrate its importance, vitamin D deficiency is associated with an increased risk for nursing home admission. The hazard ratio of nursing home admission after 6 years of follow-up for vitamin D-deficient individuals (25OHD₃ < 25 nmol/l) in a large cohort of older people was 3·48 (1·39–8·75) when compared with individuals with a high serum 25OHD₃ level. The hazard ratio for vitamin D-insufficient individuals (25OHD₃ = 25–49 nmol/l) was 2·77 (1·17–6·55)⁽Reference Visser, Deeg and Puts⁸⁷⁾. The effects of vitamin D on bone, intestine and kidney, which are regarded as the classical target tissues, have been the subject of many studies for a long period of time. However, as the VDR is being found in increasingly more tissues, implications of vitamin D in many different disease states are being reported due to the effects of vitamin D outside these classical target tissues (Fig. 2). Detailed effects of vitamin D have been reported on cardiovascular health, immune system, neurological diseases and cancer. The discussion of the effects of vitamin D in these disease states is beyond the scope of the present paper, but excellent reviews have recently been published⁽Reference Hewison^3, Reference McCann and Ames^5, Reference Richart, Li and Staessen^88, Reference Giovannucci⁸⁹⁾. Of note, recently adipose tissue has been shown to be a target tissue of vitamin D⁽Reference Duque and Troen⁹⁰⁾. With ageing, there is an accumulation of fat in bone marrow at the expense of osteoblastogenesis, contributing to the development of senile osteoporosis. Vitamin D has been shown to block adipogenesis by inhibiting the expression of PPARγ2, a critical transcription factor for adipogenesis in bone marrow⁽Reference Duque and Troen⁹⁰⁾.

Fig. 2 Consequences of ageing on both vitamin D endocrine system and calcium absorption. 25OHD₃, 25-hydroxyvitamin D₃; VDR, vitamin D receptor; 1,25(OH)₂D, 1,25-dihydroxyvitamin D; IGF, insulin-like growth factor; 24OHase, 24-hydroxylase; TRPV, transient receptor potential vanilloid; AID, auto-immune disorder.

In general, the advancing knowledge of the effects of vitamin D in all these tissues further strengthens the call for adequate treatment of vitamin D deficiency⁽Reference Kimball, Fuleihan Gel and Vieth⁹¹⁾.

Treatment of vitamin D deficiency

Given the high prevalence of vitamin D deficiency in old age and the severe health consequences, a proactive approach from clinicians to case finding and adequate treatment of vitamin D deficiency is needed. Important considerations besides age are sex, BMI, skin colour, mobility, housing and dietary intake of both Ca and vitamin D⁽Reference Holick, Matsuoka and Wortsman^35, Reference Bouillon, Verstuyf and Mathieu^92, Reference Semba, Garrett and Johnson⁹³⁾. Giving individualised treatment advice is complicated by the fact that the ideal vitamin D level has not yet been defined and the treatment effect on, for example, secondary hyperparathyroidism is also dependent on the dietary intake of Ca, which shows regional differences. In general, mobile, Caucasian community-dwelling elderly, who have a varied diet, need vitamin D supplementation of 10–20 μg (400 IU–800 IU)/d to reach serum vitamin D levels of 50–75 nmol/l. Frail or institutionalised elderly on the other hand are suggested to need up to 50 μg (2000 IU)/d⁽Reference Heaney^84, Reference Bischoff-Ferrari, Giovannucci and Willett⁹⁴⁾. The effectiveness of this high-dose vitamin D supplementation in raising serum 25OHD₃ levels adequately has been demonstrated in several clinical trials⁽Reference Himmelstein, Clemens and Rubin^95–Reference Stefikova, Chylova and Krivosikova⁹⁷⁾. However, robust evidence on the optimal dose of vitamin D supplementation in specific high-risk groups is still lacking. In a recent report by the Dutch Health Council⁽⁹⁸⁾, 20 μg (800 IU) daily is advised for high-risk groups, i.e., persons with osteoporosis, residents of care homes, women aged 50+ and men aged 70+ with dark skin colour or housebound individuals.

Oral supplementation is the most effective intervention to treat vitamin D deficiency. Ergocalciferol is equally as effective as cholecalciferol in raising serum 25OHD₃ levels⁽Reference Holick, Biancuzzo and Chen⁹⁹⁾. Daily dosing is the most efficient interval to raise serum 25OHD₃ concentrations when compared with weekly or monthly administration⁽Reference Chel, Wijnhoven and Smit²⁶⁾.

Although vitamin D supplementation therapy is generally regarded as safe, cases of iatrogenic and accidental overdose with cholecalciferol have been reported⁽Reference Blank, Scanlon and Sinks^100, Reference Hathcock, Shao and Vieth¹⁰¹⁾. Most safety data concerning the use of high-dose cholecalciferol supplementation come from observations in relatively young individuals. Few studies have used high-dose cholecalciferol supplementation for longer periods in frail, older patients. Frail old people, particularly the institutionalised, often have poor daily fluid intake, use diuretics and have less thirst sensation than younger persons.

The need for high-dose supplementation therapy on the one hand, and the increased risk of dehydration on the other hand, may potentially increase the risk of accidental hypercalcaemia in these patients.

Recently, concerns have risen regarding the possible negative health effects of vitamin D supplementation⁽Reference Richart, Li and Staessen⁸⁸⁾. The recent discovery of FGF-23 and klotho has given more insight into possible negative health effects of vitamin D supplementation and hypervitaminosis D. In animal studies, both FGF-23 and klotho knockout mice have increased expression of the enzyme 1α-OHase. These mice have increased serum levels of 1,25(OH)₂D₃, Ca and phosphate and have overall an identical phenotype. These knockout mice, despite their high levels of 1,25(OH)₂D₃, develop osteoporosis, vascular and soft tissue calcifications, muscle wasting, pulmonary emphysema and have a shortened lifespan⁽Reference Tsujikawa, Kurotaki and Fujimori^102, Reference Shimada, Kakitani and Yamazaki¹⁰³⁾. Klotho knockout mice have very high levels of FGF-23 (about 2000-fold higher), but have no sign of phosphaturia, illustrating the importance of klotho for FGF-23 signalling⁽Reference Lanske and Razzaque^18, Reference Urakawa, Yamazaki and Shimada²¹⁾. Normalisation of vitamin D activity in both klotho and FGF-23 knockout mice by either feeding them a vitamin D-deficient diet or knockout of the 1α-OHase enzyme increased survival and rescued most of the phenotype, illustrating that these effects are indeed vitamin D related⁽Reference Tsujikawa, Kurotaki and Fujimori^102, Reference Razzaque and Lanske¹⁰⁴⁾. Similar effects of hypervitaminosis D due to accidental overdose in human subjects have been reported⁽Reference Hathcock, Shao and Vieth^101, Reference Jacobus, Holick and Shao¹⁰⁵⁾. Discontinuation of vitamin D supplementation in human subjects with hypervitaminosis D due to excessive intake of vitamin D resulted in normalisation of serum levels of 25OHD₃, gradual recovery of bone density mineral and normalisation of the ratio of urinary Ca to creatinine⁽Reference Adams and Lee¹⁰⁶⁾. So, when a patient is suffering from osteoporosis, clinicians should also consider, although rare, the possibility of vitamin D overdose.