Calcidiol hydroxylases

The precursor, 7-dehydrocholesterol is converted by a non-enzymatic reaction to cholecalciferol (calciol). This reaction occurs in skin exposed to sunlight due to irradiation by UV-B light at a wavelength of about 300 nm. Cholecalciferol is transported via carrier proteins to the liver where hydroxylation at carbon-25 occurs in a reaction catalysed by a microsomal cytochrome P450 hydroxylase to form calcidiol. This compound travels to the kidney attached to specific binding proteins, where another cytochrome P450 enzyme, mitochondrial 1-a-hydroxylase, introduces a second hydroxyl group in to the molecule to form the active calcitriol. 

In addition to 1-a-hydroxylase, the kidney also possesses a 24-hydroxylase which uses calcidiol as substrate the product of the reaction, 24,25 dihydroxy D3, is biologically inactive. This represents an important control point in the pathway. The activity of the 1-a-hydroxylase is promoted by calcium ions and the action of PTH acting via a G-protein/cAMP cascade. However, calcitriol itself simultaneously induces the 24-hydroxylase and suppresses 1-a-hydroxylase creating an effective feedback loop (Figure 8.12). 

Cholecalciferol 25-hydroxylase is not restricted to the liver kidneys, skin, and gut microsomes also have a cytochrome P450 -dependent enzyme that catalyzes the 25-hydroxylation of cholecalciferol and la-hydroxycholecalciferol, hut not ergocalciferol. Although there is some evidence that calcitriol can reduce the activity of calciferol 25-hydroxylase, it is not known whether this is physiologically important the major factor controlling 25-hydroxylation is the rate of uptake of cholecalciferol into the liver. It is the fate of calcidiol in the kidneys that provides the most important regulation of vitamin D metabolism (Wikvall, 2001). 

The active metabolite of vitamin D, calcitriol, is formed in the proximal tubules of the kidneys from calcidiol. There are three cytochrome P450-dependent enzymes in kidneys that catalyze 1-hydroxylation of calcidiol CYP27A and CYP27 in mitochondria and a microsomal la-hydroxylase, which is ferredoxin-dependent. It is likely that the microsomal enzyme is the most important its synthesis is induced by cAMP in response to parathyroid hormone (Section 3.2.8.2) and repressed by calcitriol (Omdahl et al., 2001 Wikvall, 2001). 

Calcidiol la-hydroxylase is not restricted to the kidney, but is also found in placenta, bone cells (in culture), mammary glands, and keratinocytes. The placental enzyme makes a significant contribution to fetal calcitriol, but it is not clear whether the calcidiol 1-hydroxylase activity of other tissues is physiologically significant or not. Acutely nephrectomized animals given a single dose of calcidiol do not form any detectable calcitriol, but there is some formation of calcitriol in anephric patients, which increases on the administration of cholecalciferol or calcidiol. However, thus extrarenal synthesis is not adequate to meet requirements, so that osteomalacia develops in renal failure (Section 3.4.1). The enzyme is inhibited, or possibly repressed, by strontium ions this is the basis of strontium-induced vitamin D-resistant rickets, which responds to the administration of calcitriol or la-hydroxycalciol, but not calciferol or calcidiol (Omdahl and DeLuca, 1971). 

Calcidiol la-hydroxylase also acts on 24-hydroxycalcidiol, yielding cal-citetrol indeed, it has a relatively low specificity and will act on any secosteroid with hydroxyl groups at C-3 and C-25. Calcitriol has a short metabolic half-life after injection of the order of 4 to 6 hours (Holick, 1990). But, under normal conditions, the regulation of its synthesis means that the plasma concentration remains fairly constant, depending on the state of calcium balance (Hewison et al., 2000). 

Both calcidiol and calcitriol are substrates for24-hydroxylation, catalyzed by a cytochrome P4so-dependent enzyme in kidneys, intestinal mucosa, cartilage, and other tissues that contain calcitriol receptors. This enzyme is induced by calcitriol the activities of calcidiol 1-hydroxylase and 24-hydroxylase in the kidney are subject to regulation in opposite directions, so that decreased requirement for, and synthesis of, calcitriol results in increased formation of 24-hydroxycalcidiol. Kidney epithelial cells in culture show increased formation of 24-hydroxycalcidiol, and decreased formation of calcitriol, after the addition of calcitriol or high concentrations of calcium to the culture medium. 

Conversely, the addition of parathyroid hormone results in decreased 24-hydroxylation and increased 1-hydroxylation (Juan and DeLuca, 1977 Omdahl et al 2001 Wikvall, 2001). There is evidence that the high prevalence of vitamin D deficiency among people from the Indian subcontinent may he because of genetically determined high activity of calcidiol 24-hydroxylase, rather than cultural and dietary factors (Awumey et al., 1998). 

There is evidence that 24-hydroxycalcidiol has physiological functions distinct from those of calcitriol, and the regulation of the 24-hydroxylase suggests that it functions to provide a metabolically active product, as well as diverting calcidiol away from calcitriol synthesis (Henry, 2001). Studies of knockout mice lacking the 24-hydroxylase show that 24-hydroxycalcidiol has a role in both in-tramembranous bone formation during development and the suppression of parathyroid hormone secretion (St-Arnaud, 1999 van Leeuwen et al., 2001). 

Compounds that induce cytochrome P450-dependent hydroxylases, such as barbiturates and the anticonvulsants primidone and diphenylhydantoin, cause increased output of vitamin D metabolites in the bile, and increase the rate of inactivation of calcidiol by liver microsomes. As a result of this, longterm use of these anticonvulsants may be associated with the development of... 

Calcitriol The major determinant ofthe relative activities of calcidiol 1 -hydroxylase and 24-hydroxylase is the avaUahUity of calcitriol. In vitamin D-deficient animals, with low circulating concentrations of calcitriol, the activity of 1 -hydroxylase in the kidneys is maximal. There is litde or no detectable 24-hydroxylase activity. Both in vivo and in isolated kidney cells in culture, the addition of calcitriol results in induction of the 24-hydroxylase and repression of 1 -hydroxylase removal of calcitriol from the culture medium results in induction of 1-hydroxylase and repression of 24-hydroxylase. 

Calcitriol acts like a steroid hormone, binding to, and activating, nuclear receptors that modulate gene expression. More than 50 genes are known to be regulated by calcitriol (see Table 3.3), but vitamin D response elements have only been identified in a relatively small number, including calcidiol 1-hydroxylase and 24-hydroxylase calbindin, a calcium binding protein in the... 

Strontium intoxication can cause vitamin D-resistant rickets because strontium is a potent inhibitor of calcidiol 1-hydroxylase (Omdahl and DeLuca, 1971). 

Renal failure is associated with an osteomalacia-like syndrome, renal osteodystrophy, as a result of the loss of calcidiol 1-hydroxylase activity. The condition may be complicated by defective reabsorption of calcium and phosphate from the urine. Furthermore, the half-life of parathyroid hormone is increased, because the principal site of its catabolism is the kidney, so there is increased parathyroid hormone-stimulated osteoclastic action without the compensatory action of calcitriol (Mawer etal., 1973). 

Hypoparathyroidism is also associated with a failure of calcidiol 1-hydroxylation, in this case because the major stimulus for induction of 1-hydroxylase is parathyroid hormone. 

X-linked hypophosphatemic rickets is caused by abnormal reabsorption of phosphate in the proximal renal tubule, resulting in excessive excretion of phosphate and hence hypophosphatemia. There may also be blunting of the normal increase in calcidiol 1 -hydroxylase activity in response to hypophosphatemia. The gene responsible for the condition has been identified (the PHEX gene) its product is a membrane-bound endopeptidase that normally acts to clear the hormone phosphatonin from the circulation. Phosphatonin acts to decrease the activity of the sodium/phosphate cotransporter in the kidney (Drezner, 2000). 

Type 1 vitamin D-resistant rickets is due to a genetic defect in calcidiol 1-hydroxylase, so that litde or no calcitriol is formed. Patients respond well to the administration of la-hydroxycholecalciferol, which is a substrate for 25-hydroxylation in the liver, leading to normal circulating concentrations of calcitriol. 

Plasma Concentrations of Calcium and Phosphate Although the mean response to changes in plasma cedcium is a chemge in the secretion of peuathyroid hormone, the activity of calcidiol 1-hydroxyleise in kidney slices is decreased directly by high concentrations of cedcium in the incubation medium. Cedcium heis no direct effect on the activity of calcidiol 24-hydroxylase under these conditions. Strontium and cadmium edso inhibit ctdcidiol 1-hydroxylase. 

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