Essay: Vitamin D3

Background
Vitamin D3, a fat-soluble steroid hormone known as cholecalciferol, is critical for calcium homeostasis and bone metabolism [1]. Vitamin D3 acts by enhancing calcium absorption and mobilizing calcium from bone, thus helping to build and maintain strong bones and teeth. Endogenous vitamin D is not biologically active, and instead must be metabolized within the body into its active form. Due to its hydrophobicity, active vitamin D3 must be transported in the blood using primarily the vitamin D-binding protein (VDP) in order to reach its target cells containing the vitamin D receptor (VDR) [1]. Similar to other steroid hormone receptors, the VDR has a DNA binding domain and hormone-binding domain. In order to bind DNA, the VDR forms a heterodimer with the retinoid X receptor [2]. Through binding of vitamin D3, VDR acts as a transcription factor for gene expression, resulting in a subsequent increase in calcium levels in order to maintain calcium homeostasis [1].
Calcium homeostasis, or the regulation of the concentration of calcium, is tightly controlled. Under normal circumstances the plasma calcium concentration remains constant, however, in disease states, the calcium concentration can become too high (hypercalcemia) or too low (hypocalcemia). During the 1950’s in Great Britain, an outbreak in hypercalcemia in infants was proposed to be a consequence of the over fortification of milk with vitamin D for the prevention of rickets [3]. Initially, it was thought that nutritional vitamin D intake was responsible for the pathogenesis of the disorder. However, many of the infants receiving the prophylaxis with vitamin D remained unaffected, and thus it was proposed that there were other contributing intrinsic factors [3]. Following the outbreak, the disorder was determined to be Idiopathic Infantile Hypercalcemia (IIH).
Infants with IIH experience vomiting, increased urination, dehydration, constipation, weight loss, and an inability to grow and gain weight normally [3]. Individuals with IIH may also have high levels of calcium in the urine (hypercalciuria) or deposits of calcium in the urine (nephrocalcinosis).
Overview of Normal Vitamin D Metabolism and Calcium Homeostasis
The primary sources of vitamin D include exposure to sunlight, which is necessary for ultraviolet-B-induced vitamin D production within the skin [4], along with diet and supplementation. Once in the body, vitamin D3 undergoes activation through several hydroxylation reactions (Fig.1.). First, vitamin D3 is hydroxylated by 25-hydroxylase (CYP2R1) in the liver to produce the primary circulating form, 25-hydroxyvitamin D3 [3]. Next, a second hydroxylation reaction catalyzed by the enzyme 1a-hydroxylase (CYP27B1) in the kidney results in the biologically active form 1,25-dihydroxy vitamin D3 [3]. When levels of 1,25-(OH)2 D3 are high, it is then inactivated through catabolism by 24-hydroxylase (CYP24A1) into 24,25-(OH)2D3 (calcitroic acid) primarily [3].
Figure 1. Vitamin D Metabolism Under Healthy Conditions. Vitamin D is activated through several subsequent hydroxylation reactions. Once active, vitamin D3 can exert its biological effect through binding to the vitamin D receptor (VDR) and is then catabolized by 24-hydroxylase into products that are more readily excreted.
Calcium homeostasis is regulated through a negative feedback hormone system, the two most important hormones being parathyroid hormone and 1,25(OH)2D3. PTH is the major regulator of extracellular calcium concentration. When serum calcium is low, the calcium-sensing receptor (CaR) in the parathyroid gland becomes inactivated, and as a result, increases PTH secretion from the parathyroid gland [5]. PTH acts on the PTH receptor in the kidney and the bone by stimulating calcium reabsorption and bone resorption respectively. PTH also indirectly stimulates calcium absorption in the gut by triggering the synthesis of 1,25-(OH)2D3 in the kidney [5]. This restores levels of calcium, helping to maintain a baseline extracellular calcium level.
The Power of Animal Models
It is widely known that vitamin D influences bone metabolism indirectly through the maintenance of calcium and phosphate homeostasis. Recent studies have revealed “direct, but non-essential roles for 1,25-(OH)2D in growth plate chondrocytes” [6]. In order to test the hypothesis that 1,25-(OH)2D plays a direct role in chondrocytes, a mouse model was developed allowing for specific inactivation of CYP27B1 genes. The resulting decrease in 1,25-(OH)2D and thus inactivation of the vitamin D receptor (VDR) in chondrocytes was found to result in delayed osteoclastogenesis and thus an increase in bone volume [6]. The inactivated VDR mice also experienced reduced levels of FGF-23 and a resulting elevated serum phosphate concentration. Thus, it was proposed that a “1,25-(OH)2D-induced secreted factor from chondrocytes that affects FGF-23 production of neighboring osteoblasts” [6].
CYP24A1: Biochemistry, catalytic properties, physiological role, and regulation.
CYP24A1 (24-hydroxylase) catalyzes the conversion of 25-OH D3 and its hormonal form 1,25-(OH)2D3 into 24-hydroxylated products that are more easily excreted. The hydroxylation reaction catalyzed by 24-hydroxylase occurs at carbons C-24 or C-23 of the side chain [7]. CYP24A1 is said to be responsible for the 5-step 24-oxidation pathway from 1,25-(OH)2D3 to calcitroic acid, while also catalyzing a similar pathway beginning with 23-hydroxylation instead. This results in the product 1,25-(OH)2D3-26-23-lactone [7]. Moreover, studies have shown that 24-hydroxylase is expressed in numerous target cells containing the vitamin D receptors [7], which led to the proposal that the critical role of CYP24A1 is to limit the activity of active vitamin D on target cells following transcription. Catabolism is stimulated through a negative feedback mechanism, in which increased 1,25-(OH)2D3 activity is thought to trigger CYP24A1 mediated catabolism. This prevents excess activation of VDR and the resulting biological effects. This was further confirmed by the finding of two vitamin D response elements in the promotor of the CYP24A1 gene, explaining the role 1,25-(OH)2D3 has in direct regulation [8]. Although these findings suggest CYP24A1 to be the main enzyme responsible for vitamin D catabolism, it has also been found to work together with CYP27B1 [9], which is responsible for converting 25-OH D3 into its biologically active form.
In addition to being regulated by the activity of 1,25-(OH)2D3, CYP24A1 is also regulated by the parathyroid hormone (PTH) and FGF-23 [7]. Firstly, PTH plays a crucial role in regulating calcium-phosphate metabolism. When serum calcium levels are low, PTH is produced more extensively. PTH stimulates CYP27B1 expression thereby converting vitamin D3 into its active form, 1,25-(OH)2D3 [7]. The active form of vitamin D can the bind VDR inducing calcium absorption to help correct the low levels of calcium. In the kidney, 1,25-(OH)2D3 mediated induction of CYP24A1 is weakened when PTH is high. This is due to destabilization and increased degradation of CYP24A1 mRNA, which is achieved through the cAMP/PKA signaling pathway [7]. To contrast, in bone cells, PTH stimulates 1,25-(OH)2D3 induction of CYP24A1 transcription through the cAMP-signaling pathway [7]. That is to say that PTH induction of CYP24A1 occurs in the bone cells, and PTH suppression of CYP24A1 occurs in the kidney. The suppression of CYP24A1 in the kidney ultimately results in an increase in active vitamin D and thus an increase in calcium in order to maintain homeostasis.
FGF-23 is a bone-derived hormone that also affects the expression of genes regulating both phosphate and vitamin D metabolism. FGF-23 expression is stimulated following increased phosphate levels acting to reduce phosphate reabsorption. This is done directly by inhibiting the Na/Pi co-transporter and indirectly by reducing phosphate absorption by decreasing expression of CYP27B1, subsequently leading to decreased levels of 1,25-(OH)2D3 [7]. FGF-23 is also able to control vitamin D levels by increasing the expression of CYP24A1 mRNA in the kidney [7]. As such, FGF-23 is able to regulate levels of vitamin D by acting on both CYP27B1 and CYP24A1.
CYP24A1 and its role in the Pathophysiology of Idiopathic Infantile Hypercalcemia
Idiopathic infantile hypercalcemia is characterized by high levels of calcium in the blood. Until recently, IIH had no gene locus assigned to it and the catabolic mechanism of vitamin D via CYP24A1 remained unknown. In 2011, Schlingmann et al. demonstrated that loss-of-function mutations in CYP24A1 may be responsible for the pathogenesis [3]. Through the use of a mammalian expression system, they were able to identify and evaluate mutations in IIH patients. The results of their study revealed autosomal-recessive mutations in the IIH patients, along with other CYP24A1 mutations in infants who had developed hypercalcemia following administration of vitamin D [3]. These findings highlight the catabolic nature of CYP24A1 and lead to an understanding that the presence of loss-of-function mutations is what caused vitamin D sensitivity in patients suffering from IIH.
In 2016, Schlingmann et al. identified four patients with IIH without mutations in CYP24A1. Instead, a second IIH gene locus on chromosome 5q35, most likely being SLC34A1 [10]. Following sequence analysis of SLC34A1, which encodes the renal sodium-phosphate transporter 2A (NaPi-IIa), it was revealed that there were autosomal-recessive mutations in each case. As such, SLC34A1 may also contribute to the pathogenesis of IIH and early differentiation between the two defects will remain crucial for more targeted therapy.
Diagnostic Tools and Screening for IIH
In order to confirm a diagnosis of IIH, genotyping of the CYP24A1 gene remains critical. However, improved screening tests have recently been implemented to predict the presence of loss-of-function CYP24A1 mutations. Kaufmann et al. (2017) proposed a screening test that employed rapid liquid-chromatography tandem mass spectrometry (LC-MS/MS)-based assay [11]. This assay measures the concentration of numerous vitamin D metabolites simultaneously, providing a 25-OH D3 to 24,25-(OH)2D3 ratio. 24,25-(OH)2D3is the main product of catabolism via CYP24A1 and is thus representative of its enzymatic activity. The ratio is much more definitive that simply measuring the levels of inactivated vitamin D3, as it eliminates the possibility that the results are due to a vitamin D deficiency. This ratio, which is normally 5-25, rises to values higher than 80 for patients with IIH due to the loss-of-function CYP24A1 mutations.
The new screening assay for IIH has been 100% successful in predicting mutations which are later deemed to be recessive [11]. Despite the success, the assay still detects residual amounts of the inactive form 24,25-(OH)2D3 [11]. This may be due to another CYP isoform that retains 24-hydroxylase activity or perhaps the analyzed peak contains small traces of other vitamin D metabolites.
Current and Future Treatment Options
Hypercalcemia causes severe dehydration, and thus rehydration is often incorporated into treatment. Management of hypercalcemia also includes bisphosphonates, steroids, or calcitonin [12]. In severe cases where corticosteroids or furosemides fail, bisphosphonates can be considered. Bisphosphonates are considered last due to their side effect profile and warnings against long-term use. Despite limited data on bisphosphonate in children, Skalova et al. (2013) proved intravenous administration of pamidronate to be efficacious in an infant with severe hypercalcemia due to a CYP24A1 loss of function mutation [13]. Pamidronate, and other bisphosphonates inhibit bone resorption and proven to lower serum calcium levels [13]. Although lowering serum calcium levels, there has been concern about over suppression of bone leading to fractures after continuous use.
More specific to IIH due to CYP24A1, current treatment approaches involve limiting sunlight exposure and avoiding dietary vitamin D, reflecting the pathophysiology of the disease [12]. Since the discovery of CYP24A1 mutations in patients with idiopathic infantile hypercalcemia, other successful treatment options have been discovered to effectively control hypercalcemia, without the need for short-term steroid use. Some treatments focus on inhibiting the activation of vitamin D. This includes the general azole-based cytochrome inhibitors, ketoconazole and fluconazole. Ketoconazole is a P450 inhibitor, such as those involved in vitamin D metabolism and has been investigated as a potential therapy for the prevention of hypercalcemia [14]. Although deemed effective in reducing serum calcium through directly targeting 1,25(OH)2D3 levels, the long-term use of ketoconazole is not advisable due to the high risk of hepatotoxicity. A less toxic azole agent, fluconazole, has also been demonstrated to be successful in the treatment of IIH [14]. Unlike ketoconazole, fluconazole did not cause hepatotoxicity and therefore is hypothesized to be a more appropriate long-term treatment. It is worth noting that this study cannot predict long-term outcomes such as intolerance and future hepatoxicity [14]. Rather than inhibiting activation of vitamin D, Hawkes et al. (2017) discovered an alternate pathway for inactivation of vitamin D. Rifampin, a CYP3A4 inducer, led to normalization of vitamin D metabolites, serum calcium, and urinary calcium [12]. Therefore, induction of CYP3A4 could serve as a potential treatment for patients who lack CYP24A1 function.