The active form of vitamin D is its 1,25-dihydroxy metabolite
By the 1920s, investigators recognized that dietary deficiency of a fat-soluble vitamin was responsible for the childhood disease rickets (Box 52-2). This disorder is characterized clinically by hypocalcemia and multiple skeletal abnormalities. Dietary replacement of vitamin D corrects this disorder and has led to the practice of adding vitamin D to milk, bread, and other products. This practice has greatly reduced the prevalence of this previously common disorder.
Rickets and Osteomalacia
Deficiency of vitamin D in children produces the disease rickets, in which bone has abnormal amounts of unmineralized osteoid. Both cortical and trabecular bone are involved. The lack of mineralization diminishes bone rigidity and leads to a characteristic bowing of the long bones of the legs of affected children. In adults, vitamin D deficiency produces a disorder called osteomalacia. Microscopically, the bone looks very much the same in adult and childhood vitamin D deficiency. However, because the longitudinal growth of the long bones has been completed in adults, bowing of weight-bearing bones does not occur. Instead, the increased unmineralized osteoid content of bone causes a decline in bone strength. Affected individuals are more prone to the development of bone fractures. These fractures may be very small and difficult to see radiographically. As more and more of the bone surface is covered by osteoid and as recruitment of new osteoclasts is diminished, osteoclastic bone resorption is impaired and hypocalcemia develops. Hypocalcemia causes nerves to become more sensitive to depolarization. In sensory nerves, this effect leads to sensations of numbness, tingling, or burning; in motor nerves, it leads to increased spontaneous contractions, or tetany.
Although rickets and osteomalacia are very uncommon in developed countries because of vitamin D supplementation, milder degrees of vitamin D deficiency are recognized increasingly in the elderly population, in whom milk consumption and sunlight exposure are frequently inadequate. The resulting fall in plasma [Ca2+] can lead to mild secondary hyperparathyroidism. Such continuous elevations of PTH can lead to further bone resorption and worsening osteoporosis.
Rickets or osteomalacia also can occur with impaired ability of the kidney to 1-hydroxylate the 25-hydroxyvitamin D previously synthesized in the liver. An acquired impairment is seen in many patients with chronic renal failure, in which the activity of 1α-hydroxylase is reduced. The genetic form of the 1α-hydroxylase deficiency is a rare autosomal recessive disorder. Either form is called vitamin D–dependent rickets because it can be successfully treated with either 1,25-dihydroxyvitamin D or doses of dietary vitamin D2 or D3 that are higher (by ~10- to 100-fold) than the 400 U/day used to prevent nutritional rickets.
Vitamin D exists in the body in two forms, vitamin D3 and vitamin D2 (Fig. 52-9). N52-2 Vitamin D3 can be synthesized from the 7-dehydrocholesterol that is present in the skin, provided sufficient ultraviolet light is absorbed. This observation explains why nutritional rickets was more prevalent in northern countries, where people have reduced skin exposure to sunlight. Vitamin D3 is also available from several natural sources, including cod and halibut liver, eggs, and fortified milk. Vitamin D2 is obtained only from the diet, largely from vegetables. Vitamin D3 (see Fig. 52-9A) and vitamin D2 (see Fig. 52-9B) differ only in the side chains of ring D. The side chain in vitamin D3 (cholecalciferol) is characteristic of cholesterol, whereas that of vitamin D2 (ergocalciferol) is characteristic of plant sterols.
FIGURE 52-9 Forms of vitamin D. UV, ultraviolet. N52-2
Metabolism of Vitamin D3 and D2
Contributed by Eugene Barrett
Vitamin D3—or cholecalciferol—actually can be thought of as a hormone because it can arise entirely from the metabolism of an endogenous source (7-dehydroxycholesterol) and because it acts through a specific receptor. Ultraviolet light triggers the cleavage in the skin of the B ring of 7-dehydroxycholesterol, creating an unstable intermediate that—over a period of about 2 days—rearranges to form cholecalciferol (vitamin D3). Vitamin D3 can also come from animal sources in the diet. However, vitamin D3 is not active as such. In the liver, a P-450 enzyme hydroxylates vitamin D3 at the 25 position, creating 25-hydroxyvitamin D3. Then in the proximal-tubule cells of the kidney, another P-450 enzyme hydroxylates 25-hydroxyvitamin D3 at position 1, forming 1,25-dihydroxyvitamin D3, the active form of vitamin D3.
Vitamin D2, which comes exclusively from dietary plant sources, differs from vitamin D3 only in the side chain off carbon 17 in ring D. Like vitamin D3, vitamin D2 undergoes 25-hydroxylation in the liver and 1-hydroxylation in the kidney. Also like vitamin D3, the 1,25-dihydroxylated metabolite of vitamin D2 is about 1000-fold more active than the 25-monohydroxylated form.
Vitamin D (i.e., either D2 or D3) is fat soluble, but water insoluble. Its absorption from the intestine depends on its solubilization by bile salts (see p. 933). In the circulation, vitamin D is found either solubilized with chylomicrons (see pp. 932–933) or associated with a vitamin D–binding protein. Most of the body stores of vitamin D are located in body fat. The body's pools of vitamin D are large, and only 1% to 2% of the body's vitamin D is turned over each day. Therefore, several years of very low dietary intake (as well as diminished endogenous synthesis) are required before the endogenous pools are depleted and deficiency develops.
The principal active form of vitamin D is not vitamin D2 or D3, but rather a dihydroxylated metabolite of either. Hydroxylation of vitamin D proceeds in two steps (see Fig. 52-9A). When circulating levels of 25-hydroxyvitamin D are low, adipocytes release vitamin D into the blood plasma. A cytochrome P-450 mixed-function oxidase, principally in the liver, creates the first hydroxyl group at carbon 25. The 25-hydroxylation of vitamin D does not appear to be highly regulated, but rather it depends on the availability of vitamin D2 or D3. The second hydroxylation reaction occurs in the renal proximal tubule under the tight control of PTH, vitamin D itself, and FGF23 (see p. 787). PTH stimulates this 1-hydroxylation, whereas FGF23 and 1,25-dihydroxyvitamin D (the reaction product) both inhibit the process (see Fig. 52-8).
In addition to vitamins D2 and D3 and their respective 25-hydroxy and 1,25-dihydroxy metabolites, >15 other metabolites of vitamin D have been identified in plasma. However, the specific physiological function of these metabolites, if any, is unclear.
Although considered a vitamin because of its dietary requirement, vitamin D can also be considered a hormone, both because it is endogenously synthesized and because even the fraction that arises from the diet must be metabolized to a biologically active form.
Vitamin D and its metabolites, like the steroid hormones, circulate bound to a globulin, in this case a 52-kDa vitamin D–binding protein. This binding protein appears particularly important for carriage of vitamins D2 and D3 in plasma because they are less soluble than their hydroxylated metabolites. Vitamin D and its metabolites arrive at target tissues and, once in the cytosol, associate with the VDR, a transcription factor that is in the family of nuclear receptors (see pp. 71–72). Like the thyroid hormone receptor (see Table 3-6), VDR forms a heterodimer with RXR. The VDR specifically recognizes the 1,25-dihydroxyvitamin D with an affinity that is three orders of magnitude higher than that for 25-hydroxyvitamin D. However, because the circulating concentration of 25-hydroxyvitamin D is ~1000-fold higher than that of 1,25-dihydroxyvitamin D, both species probably contribute to the biological actions of the hormone.
The biological actions of 1,25-dihydroxyvitamin D appear to be expressed principally, but not exclusively, via regulation of the transcription of a variety of proteins. The VDR/RXR complex associates with a regulatory site in the promoter region of the genes coding for certain vitamin D–regulated proteins. Thus, the occupied VDR alters the synthesis of these vitamin D–dependent proteins. An example is PTH, which stimulates the formation of 1,25-dihydroxyvitamin D. The 5′ regulatory region of the PTH gene has a VDR consensus sequence; when occupied by the VDR complex, this element diminishes transcription of the PTH gene.
Vitamin D, by acting on the small intestine and kidney, raises plasma [Ca2+] and thus promotes bone mineralization
The actions of vitamin D can be grouped into two categories: actions on classic target tissues involved in regulating body mineral and skeletal homeostasis, and a more general action that regulates cell growth. The actions of vitamin D on the small intestine, bone, and kidney serve to prevent any abnormal decline or rise in plasma [Ca2+].
In the duodenum, 1,25-dihydroxyvitamin D increases the production of several proteins that enhance Ca2+ absorption. Figure 52-10A summarizes the intestinal absorption of Ca2+, which moves from the intestinal lumen to the blood by both paracellular and transcellular routes (see p. 938). In the paracellular route, which occurs throughout the small intestine, Ca2+ moves passively from the lumen to the blood; 1,25-dihydroxyvitamin D does not regulate this pathway. The transcellular route, which occurs only in the duodenum, involves three steps. First, Ca2+ enters the cell across the apical membrane via TRPV6 Ca2+channels (see p. 938). Second, the entering Ca2+ binds to several high-affinity binding proteins, particularly calbindin. These proteins, together with the exchangeable Ca2+ pools in the RER and mitochondria, effectively buffer the cytosolic Ca2+ and maintain a favorable gradient for Ca2+ entry across the apical membrane of the enterocyte. Thus, the intestinal cell solves the problem of absorbing relatively large amounts of Ca2+ while keeping its free cytosolic [Ca2+] low. Third, the enterocyte extrudes Ca2+ across the basolateral membrane by means of both a Ca pump and an Na-Ca exchanger.
FIGURE 52-10 Intestinal absorption of Ca2+ and phosphate. A, The small intestine absorbs Ca2+ by two mechanisms. Passive paracellular absorption of Ca2+ occurs throughout the small intestine. This pathway is the predominant one but is not under the control of vitamin D. The second mechanism—active transcellular absorption of Ca2+—occurs only in the duodenum. Ca2+ enters the cell across the apical membrane via TRPV6 Ca2+ channels. Inside the cell, the Ca2+ is buffered by binding proteins, such as calbindin, and is also taken up into intracellular organelles, such as the ER. The enterocyte then extrudes Ca2+ across the basolateral membrane via a Ca pump and an Na-Ca exchanger. Thus, the net effect is Ca2+ absorption. The active form of vitamin D—1,25-dihydroxyvitamin D—stimulates all three steps of transcellular Ca2+ absorption. B, Inorganic phosphate (Pi) enters the enterocyte across the apical membrane via an Na/Pi cotransporter (NaPi). Once inside the cell, the Pi is extruded across the basolateral membrane. Thus, the net effect is Pi absorption.
Vitamin D promotes intestinal Ca2+ absorption primarily by genomic effects that involve induction of the synthesis of epithelial Ca2+ channels and pumps and Ca2+-binding proteins, as well as other proteins (e.g., alkaline phosphatase).
The effect of PTH to stimulate intestinal Ca2+ absorption is thought to be entirely indirect and mediated by PTH's action to increase the renal formation of 1,25-dihydroxyvitamin D (see Fig. 52-8), which then enhances intestinal Ca2+ absorption.
Vitamin D also stimulates phosphate absorption by the small intestine (see Fig. 52-10B). The initial step—as in the renal proximal tubule (see pp. 785–786)—is mediated by the NaPi cotransporter and appears to be rate limiting for transepithelial transport and subsequent delivery of phosphate to the circulation. 1,25-Dihydroxyvitamin D stimulates the synthesis of this transport protein and thus promotes phosphate entry into the mucosal cell.
In the kidney, vitamin D appears to act synergistically with PTH to enhance Ca2+ reabsorption in the DCT (see p. 789). High-affinity Ca2+-binding proteins, similar to those found in the intestinal mucosa, have been specifically localized to this region of the kidney. It appears that PTH is a more potent regulator of Ca2+ reabsorption than is vitamin D (see Fig. 52-8). Indeed, parathyroidectomy increases the fractional excretion of Ca2+, and even high doses of vitamin D cannot correct this effect. In addition, as in the intestine, vitamin D promotes phosphate reabsorption in the kidney. The effects of vitamin D on phosphate reabsorption, like its effects on Ca2+, are less dramatic than those of PTH. Finally, 1,25-dihydroxyvitamin D directly inhibits the 1-hydroxylation of vitamin D, establishing a negative-feedback loop.
The actions of vitamin D on bone are complex and are the result of both indirect and direct actions. The overall effect of vitamin D replacement in animals with diet-induced vitamin D deficiency is to increase the flux of Ca2+ into bone. However, as we see below, the major effects of vitamin D on bone are indirect: the action of vitamin D on both the small intestine and the kidneys makes more Ca2+ available to mineralize previously unmineralized osteoid. The direct effect of vitamin D on bone is via both osteoblasts and osteoclast precursor cells, both of which have VDRs. Vitamin D increases both osteoblastic and osteoclastic differentiation; when these activities are balanced, vitamin D simply increases bone turnover. However, when vitamin D is present in excess, it favors bone resorption because osteoblasts produce certain proteins with matrix-destroying properties (e.g., alkaline phosphatase, collagenase, plasminogen activator) as well as proteins that favor osteoclastogenesis (e.g., RANKL rather than OPG; see Fig. 52-4). Thus, because vitamin D can directly increase the number of mature osteoclasts, supplying vitamin D to bone obtained from vitamin D–deficient animals in in vitro experiments mobilizes Ca2+ from bone into the medium. Additional evidence that vitamin D directly promotes bone resorption comes from experiments on rachitic animals who are maintained on a calcium-deficient diet. Treating these animals with vitamin D causes plasma [Ca2+] to rise, an indication of net bone resorption. At the same time, however, the elevated plasma [Ca2+] promotes the mineralization of previously unmineralized osteoid—at the expense of bone resorption from other sites.
In summary, the antirachitic action of vitamin D is both indirect and direct. By enhancing the absorption of Ca2+ and phosphate from the intestine and by enhancing the reabsorption of Ca2+ and phosphate from the renal tubules, vitamin D raises the concentrations of both Ca2+ and phosphate in the blood and ECF. This increase in the Ca × PO4 ion product, along with more differentiated osteoblasts, results—indirectly—in net bone mineralization. On the other hand, when vitamin D is in excess, the direct effect of vitamin D predominates, increasing bone mobilization.
Calcium ingestion lowers—whereas phosphate ingestion raises—levels of both PTH and 1,25-dihydroxyvitamin D
When an individual ingests a meal containing calcium, the ensuing rise in plasma [Ca2+] inhibits PTH secretion. The decline in PTH causes a decrease in the resorption of Caprotein 2+ and phosphorus from bone, thus limiting the postprandial increase in plasma Ca2+ and phosphate levels. In addition, the decrease in PTH diminishes Ca2+ reabsorption in the kidney and thus facilitates a calciuric response. If dietary Ca2+intake remains high, the lower PTH will result in decreased 1-hydroxylation of 25-hydroxyvitamin D, which will eventually diminish the fractional absorption of Ca2+ from the GI tract.
If dietary Ca2+ intake is deficient, the body will attempt to restore Ca2+ toward normal by increasing plasma [PTH]. This response will help to mobilize Ca2+ from bone, to promote renal Ca2+ retention, and, over time, to increase the level of 1,25-dihydroxyvitamin D, which will enhance gut absorption of Ca2+.
If one ingests phosphorus much in excess of Ca2+, the rise in plasma [phosphate] will increase the plasma Ca × PO4 ion product, thereby promoting deposition of mineral in bone and lowering plasma [Ca2+]. The low plasma [Ca2+], in turn, increases PTH secretion, provoking a phosphaturia and thus a fall of plasma [phosphate] toward normal. In addition, the PTH mobilizes Ca2+ and phosphate from bone by its action. Over longer periods, the action of PTH to modulate the 1-hydroxylation of 25-hydroxyvitamin D plays an increasingly important role in defending the plasma [Ca2+] by increasing intestinal Ca2+ absorption.
The plasma [phosphate] is thus largely maintained indirectly through the actions of PTH in response to [Ca2+]. Another regulator of plasma [phosphate] is the protein fibroblast growth factor 23 (FGF23; see p. 787), secreted by osteocytes and osteoblasts in response to high plasma [phosphate]. FGF23 acts on the intestine to decrease phosphate absorption and on the kidney to limit phosphate reabsorption. Excessive FGF23 production causes an autosomal form of hereditary hypophosphatemia rickets by impairing bone mineralization, secondary to phosphate deficiency. Conversely, FGF23 deficiency can lead to hyperphosphatemia and ectopic calcification.