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Chapter 47 The prehormone vitamin D
Chapter 47
The Prehormone Vitamin D
JAN T. DABEK
History Rickets and Osteomalacia The Chemistry of Vitamin D Dietary Requirement of Vitamin D Vitamin D Metabolism The Vitamin D Endocrine System The Cellular Receptor for 1-a,25-DihydroxyvitaminD Transcription Induced by Active 1,25-DihydroxyvitaminD Receptor Vitamin D Transport Protein Vitamin D and Classical Rickets Nomenclature of Vitamin D Compounds Some Further Aspects Renal Vitamin D Metabolism Vitamin D and the Parathyroid Glands Vitamin D Deficiency and the Parathyroid GIands Vitamin D in Relation to Certain Other Diseases Summary
HISTORY That the childhood bone deforming disease rickets could be induced by deficiency of a fat soluble substance was shown in 1919by Mellanby. While Mellanby thought Principles of Medical Biology, Volume 8C Molecular and Cellular Pharmacology,Pages 933-949. Copyright O 1997 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-813-7
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that the factor was vitamin A, McCollum showed that a new substance was involved and gave it the name vitamin D. The situation was complicated by the finding by Huldshinsky that not only fish liver oil but also ultraviolet light could in some way provide the factor. Steenbock and Black showed that even ultraviolet irradiation of food could act to induce the factor. This led to the possibility of preparing sufficient amounts of vitamin D for the structure of vitamin D2 (the plant fat form) to be determined by Askew and Windaus in the early 1930s. As a consequence of this advance in chemistry, it was possible to synthesize another active form, vitamin D3 which Schenck identified as the form of vitamin D in animal fat. In the last 20 years or so, due to the availability of highly radiolabeled but relatively stable vitamin D, other forms of the vitamin have been discovered. These include two crucial hydroxylated forms named 25-hydroxyvitamin D and 1-a, 25-dihydroxyvitamin D. The former is the main metabolite of vitamin D produced by the liver, and the latter the hormonal form produced in the kidney which is involved in calcium absorption and bone remodeling.
RICKETS A N D OSTEOMALACIA The significance of vitamin D is inextricably bound up with the history and understanding of the bone diseases rickets, in children, and osteomalacia in adults. These diseases are primarily associated with disordered bone growth, decreased bone strength, and deformity resulting from inadequatecalcification of bone matrix in cartilaginous or fibrous osteogenesis. In children the long bones do not grow normally and the epiphyseal growth plate is thickened leading, in advanced cases, to palpable nodulation near the ends of the long bones. The platelike fibrous bones, such as those of the vault of the skull, can be indented by pressure, a sign called craniotabes, and the face takes on a rectangular form. The junctions between calcified rib and costal cartilage are nodularly expanded giving rise to a sign called the rachitic rosary. The legs become bowed and the pelvis deformed by weight bearing, which in girls may later result in difficulties in parturition. In the absence of treatment, the stature remains short and after the closing of the epiphyses the deformities remain. The gait tends to be waddling due to both muscular weakness and pelvic deformity; weakness of the abdominal muscles results in abdominal distension. In the adult there is bone pain and so-called pseudofractures on X-ray (Milkman's fractures, Looser's zones), which are small areas of increased radiolucency due to markedly lessened calcification in cartilaginous bones. These are perhaps most common in the rami of the pubis and the neck of the femur. Histologically the main finding is decreased calcification of newly formed bone matrix which appears as zones of clearly undercalcified tissue along bony trabeculae. In marginal cases of vitamin D deficiency, symptoms may show exacerbations in the darker seasons, during growth spurts in children (adolescence), and during pregnancy, where the needs of the fetus take priority over those of the mother. In
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addition to the above features, advanced cases of osteomalacia have been earlier thought to show inadequate hematopoiesisand poor resistance to infection, features which may have been given a new significance in the light of recent research into the ramifying effects of hydroxylated vitamin D metabolites on cellular differentiation.
THE CHEMISTRY OF VITAMIN D Vitamin Dg or cholecalciferol (C27H440) refers to the secosteroid (a steroid with an opened ring) which can well be thought of as deriving from the steroid 7-dehydrocholesterol (Figure 1) by opening of its r ring between its ninth and tenth carbon atoms. Subsequent turning of the A-ring through 180" around bond C6-C7 makes dangles below the C- and D-rings with C3 at the lower left extreme, linked to them by the "remains" of the B-ring (Figure 2). There is one hydroxy group at C3 and three conjugated double bonds atC5-C6, C7-C8, and C 10-C19, which gives vitamin D-compounds their strong absorption at 265 nm (molar extinction coefficient about 460 per mol/l per cm). Vitamin D2 only differs as far as the side-chain of its natural precursor, ergosterol, differs from that of 7-dehydrocholesterol, i.e., it has a double bond between C22-C23 and a methyl replacing one of the hydrogens at C24. There are also other forms of vitamin D, such as vitamin D4 and D5with
Figure 7.
The structure of 7-dehydrocholesterol.
JAN T. DABEK
Figure 2. The structure of vitamin D3.
slightly differing side-chains but these are not of essential physiological interest. Ergosterol and 7-dehydrocholesterol, the natural precursors of vitamin D in plants and animals, respectively, are found in nature more widely than the vitamin forms themselves which seem mainly to be formed by exposure of the former to ultraviolet light of suitable wavelength (about 280 to 310 nm) with subsequent thermal rearrangement. Other wavelengths may destroy vitamin D by converting it to other closely related but non-vitamin compounds.
The Prehormone Vitamin D
DIETARY REQUIREMENT OF VITAMIN D Each microgram of vitamin D corresponds to 40 international units of antirachitic activity. The normal daily requirement is 200 to 400 iu or 5 to 10 pg, with the higher level during growth and development and the lower in mature adults. This can be obtained from fortified milk (usually as vitamin D2), fish liver oils (vitamin D3), butter, egg yolk, liver, or from ultraviolet light acting on skin precursors. During pregnancy and lactation a supplement of an extra 5 pg per day has been recommended. For treatment of dietary or other forms of vitamin D deficiency-hypoparathyroidism and renal osteomalacia-thousands and tens of thousands of units of the vitamin may be required or then treatment is with hydroxylated forms of the vitamin according to the latest research findings.
VITAMIN D METABOLISM On entry to the extracellular fluid space or blood, vitamin D is rapidly bound to its transport protein, vitamin D-binding globulin (DBP), and then taken up within an hour or so by the liver. The liver has two enzyme systems for hydroxylation of the vitamin at carbon-25, to give 25-hydroxyvitamin D (Figure 3), a more active microsomal mono-oxygenase and a less active mitochrondrial enzyme that may be more relevant in states of vitamin D repletion or excess. The microsomal enzyme involves a specific NADPH-cytochrome P-450 reductase which seems to be inhibited by 1,25-dihydroxyvitamin D and low or high calcium levels. The 25-hydroxyvitamin D secreted by the liver, even within a few hours of initial vitamin D uptake, is hydroxylated in the 1-aposition (the a means that the hydroxy group is below the plane of the steroid rings as normally oriented in acyclopentanophenanthrene backbone) by a renal proximal tubule hydroxylase to give 1-a,25-dihydroxyvitamin D (Figure 4). It is located in the inner part of mitochondria and is a three-component mixed-function oxidase. The functioning of this enzyme involves successive reduction along the chain NADPH, renal ferridoxin reductase (a flavoprotein loosely named renal ferridoxin), then a cytochrome P-450. The enzyme is stimulated by parathyroid hormone, though perhaps more directly by the reduced phosphate levels this hormone brings about; the prevailing calcium level may also independently modulate the enzyme, and there is some evidence for a stimulatory role for growth hormone, prolactin and calcitonin. These are the two most important hydroxylations and 1,25-dihydroxyvitamin D is regarded as the main hormonal form of vitamin D. There are also, however, many other hydroxylated forms, such as 24,25-dihydroxyvitamin D, the renal hydroxylase induced by 1,25-dihydroxyvitamin D. This 24,25-dihydroxyvitamin is active on cartilage in some stages of growth and development and it may have some longer term actions on bone, but these are poorly defined and difficult to unravel. Other hydroxylated forms are 25,26-dihydroxyvitamin D and 1,24,25-trihydroxyvitamin D. There is also a 23,25-dihydroxy- and a 23,25,26-trihydroxyvitaminD; the latter
JAN T. DABEK
Figure 3.
The structure of 25-hydroxyvitamin D3.
may transform to the 25-hydroxy,26,23-lactoneform. The hydroxylation at the 24-position is in the R-orientation but in the 23-position in the S-orientation. (Viewed from the direction of the lightest side-group at a tetrahedral carbon atom the remaining groups are arranged in clockwise (R) or anticlockwise (S) order by increasing weight or group priority according to an agreed international scheme.) 1,25-dihydroxyvitamin D is metabolized to a 23-carboxylic acid called calcitroic acid, a polar inactived form, by target cell metabolism, possibly via 24R-
The Prehorrnone Vitamin D
Figure 4.
The structure of I-a,25-dihydroxyvitarnin D3.
then 23-hydroxylations prior to side-chain cleavage. Glucuronides are also formed and secreted in bile and urine; they may be hydrolyzed in the bowel so that enterohepatic circulation of vitamin D metabolites occurs. In addition to these main hydroxylations in the liver and kidney, the placenta can also 1-a hydroxylate vitamin D thus providing an additional reserve in pregnancy. The early observations ascribing to the kidney a unique role in 1-a hydroxylation of vitamin D have been modified, particularly since vitamin D
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metabolites have emerged as paracrine, even perhaps as autocrine, factors involved in tissue differentiation and response to challenges such as cancerous disease and infection. These tissue level hydroxylations do not, perhaps, normally influence gross systemic vitamin D metabolite levels but they are important local events which, in the case of exaggerated responses, could influence gross vitamin D metabolism. Macrophages in particular have been shown to be capable of 1-a hydroxylation of vitamin D and with massive macrophage infiltration systemic 1,25-dihydroxyvitamin D levels may be raised.
THE VITAMIN D ENDOCRINE SYSTEM The conversion of vitamin D to its active hormonal form by hydroxylation of 25-hydroxyvitamin D to 1-a, 25-dihydroxyvitamin D in the kidney is indirectly under the control of the serum calcium level (Figure 5). When the serum ionized calcium level falls, the increased secretion of parathyroid hormone from the parathyroid glands either directly stimulates proximal renal tubular cells to hydroxylate more 25-hydroxyvitamin D to 1,25-dihydroxyvitaminD or accomplishes this via a phosphate lowering effect. It will be recalled that parathyroid hormone stimulates renal phosphate excretion, clearly another factor in addition to increased release of calcium from bone that raises serum and tissue fluid ionized calcium levels. As more 1,25-dihydroxyvitamin D enters the circulation more is able to accumulate in the nuclei of intestinal target cells to stimulate calcium absorption from secreted fluids and chylous food. This raises ionized calcium levels even more and eventually reduces parathyroid hormone secretion, accomplishing the aim of normalizing the ionized calcium level. As the parathyroid stimulus to the renal tubule falls off, sodoes I-a hydroxylation of 25-hydroxyvitamin D with subsequent decay of serum levels with lessening of intestinal calcium absorption. The mechanism by which 1,25-dihydroxyvitaminD acts is not yet fully understood but it is certain that it binds to a receptor protein in target cells and the complex to DNA, where it acts as a transcription factor for relevant genes such as the calcium binding proteins (Figure 6). It also seems to stimulate the activation of other genes as, for example, in cell culture where cell differentiation can be demonstrated; this indicates coordination of genetic actions by either stimulation of many genes or by taking part in a series of processes that together coordinate the action of many genes. For example, hematopoietic cell lines can be caused to differentiate along the monocyte-macrophage axis. There are cotranscription factors involved in genomic control in addition to the 1,25-dihydroxyvitaminD-receptor complex which binds to this to modulate transcription activity. Such factors are the transcription regulatory proteins which also modulate vitamin A action by complexing with receptors. The genetic actions of vitamin D require a lag phase of about two-hours, but there are also some more immediate effects such as an early entry of calcium into the enterocyte which are thought to occur perhaps by cell membrane effects. The
Parathyroids
'
ip Liver ! Vitamin D-25 hydroxylase
PTH
4 \
25-OH-D-1 hydroxylase
Bone '
I[ Figure 5. Vitamin D, its hydroxylation and hormonal control of the calcium level. 941
Figure 6. The 1,25-dihydroxyvitamin D-receptor protein complex binds to chromosomal DNA to initiate chain unwinding and transcription. Other small modulator proteins may act as co-transcription factors at various genomic sites.
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Tabk 1. Levels of Vitamin D Involvement Organism
dietary input, skin synthesis, biliary excretion, blood calcium homeostasis
Organ
intestinal calcium absorption, skeletal calcification, calcium removal from skeleton
Tissue
hydroxylated in the liver and kidney (and placenta)
Cell
binds to a receptor protein which interacts with nuclear DNA to promote transcription; has a direct effect on the cell membrane
Table 2. Physiological Effects of Vitamin D increases intestinal calcium absorption Facilitates calcium resorption from bone Facilitates bone calcification Augments intestinal phosphate transport Facilitates renal handling of calcium and phosphate Has effects on cell differentiation Influences function of endocrine glands
levels of involvement of vitamin Dare summarized in Table 1, and the major effects of 1,25-dihydroxyvitaminD are listed for convenience in Table 2.
THE CELLULAR RECEPTOR FOR 1-a, 25-DIHYDROXWITAMIN D The nuclear receptor for 1,25-dihydroxyvitamin D is a 55 kD protein located widely at low concentration in diverse cells. It is, of course, predominantly present in the major endocrine target tissues-intestine, kidney, bone-but also in the parathyroid glands, the islet cells of the pancreas, the mammary gland and many other sites as is seen in Table 3. In addition, vitamin D receptors are found in osteoclast precursors (monocytes) and also at notable levels in many cancer cells. Increased expression of the receptor for 1,25-dihydroxyvitamin D, termed up-regulation, is in part dependent on the levels of 1,25-dihydroxyvitaminD itself and up-regulation may be followed by suppression. The receptor can also be induced by glucocorticoids,which may be an important event in the neonatal period when initiation of intestinal absorption of dietary calcium is, upon complete termination of placental calcium transfer, an immediate necessity of postnatal life. The receptor has binding regions for 1,25-dihydroxyvitaminD, a region with a molecular mass of about 23,000 Da, and separated from this by a hingelike region
JAN T. DABEK
Table 3. Tissues With Vitamin D Receptors Intestinal mucosa Kidney tubule Cells of bone tissue Parathyroid Islets of Langerhans Mammary gland Keratinocytes Fibroblasts
Epithelial cells Thymocytes Lymphocytes Hematopoietic precursor cells Sertoli cells Ovarian cells Heart cells
Table 4 . The Steroid Receptor Family I. The vitamin D receptor is alike in different species. II. The following receptors are all transcription factors and similar in structure and mode of action. Vitamin D receptor Glucocorticoid receptor Retinoic acid receptor Thyroid hormone receptor Estrogen receptor
is a smaller DNA-binding stretch towards the amino end. The structure of the receptor is similar in different species. Other steroid receptors (estrogen, glucocorticoid), the thyroid hormone receptor, and a retinoic acid receptor have a clearly similar structure and these all act as gene transcription factors, as listed in Table 4. The vitamin D receptor may require phosphorylation at certain sites before it is fully competent as a transcription factor; it shows genetic polymorphism which could be related to disease patterns, e.g., osteoporosis.
TRANSCRIPTION INDUCED BY ACTIVE 1,25-DIHYDROWITAMIN D RECEPTOR Calcium binding proteins, such as calbindin-28 and calbindin-9, are induced in target cells by 1,25-dihydroxyvitamin D. These are involved in calcium transport, for example, across the bowel mucosa. Though the whole mechanism of calcium transport within cells and across cellular membranes cannot be explained by calcium binding proteins alone, the genes for these proteins have been studied and 5'-flanking promoter regions with specific palindromic nucleotide sequences responsive to the 1,25-dihydroxyvitamin D-receptor complex have been identified. Other proteins, like osteocalcin and alkaline phosphatase are also induced by hormonal vitamin D in target tissue (Table 5). This does not mean that only hormonal vitamin D elicits these responses; the same genes may be controlled by a number of factors. For example, the calcium binding protein of the uterus may be induced by estrogens.
The Prehormone Vitamin D Table 5 . Proteins Induced or Regulated by 1,25-Dihydroxyvitamin D via its Nuclear Receptor Calbindin-28 Calbindin-9 Alkaline phosphatase Osteocalcin
Ornithine decarboxylase ca2+-~~pase Renal cyclic-AMP-dependent protein kinase inhibitor
VITAMIN D TRANSPORT PROTEIN The serum DBP transports not only the vitamin in the serum but also its metabolites. It is also called vitamin D transport protein or group specific component (Gc) of which there are at least three slightly differing genetic forms all with similar vitamin binding constants. It is an albuminlike protein with an association equilibrium constant of about 5 x 10' for vitamin D. For the metabolites the associationconstants are 1 x 10' for 24,25-dihydroxyvitaminD and 1 x lo6 for 1,25-dihydroxyvitamin D. It prevents unnecessary loss of secosteroid via renal filtration and maintains a reservoir of the various metabolites ready for metabolic needs. The clearly lower affinity of this protein for 1,25-dihydroxyvitamin D facilitates the more rapid dissociation of the main hormonal form of the vitamin for speedy endocrinological effect. The level of the protein in blood plasma is about 350 mgll. In pregnancy this level may increase and thereby buffer the free concentrations of increased total serum 1,25-dihydroxyvitamin D hormone downwards. The molecular weight of DBP, which is secreted by the liver, is about 54-56,000 and the molecule contains about 6% carbohydrate. Recent DNA-level research has revealed the genes for vitamin D binding protein and the 1,25-dihydroxyvitaminD receptor protein. A virtually full-length cDNA for human serum vitamin D transport protein has been obtained from human liver messenger RNA using an expression library. Complete sequencingyielded the code for 458 amino acid residues with a calculated molecular weight of 51,335. The amino acid sequence shows strong homology with human serum albumin and a-fetoprotein.
VITAMIN D AND CLASSICAL RICKETS Without adequate serum levels of 1,25-dihydroxyvitaminD it is not possible for homeostatic mechanisms to maintain the serum calcium level (about 2.5 rnmolA) relatively constant. This is because intestinal calcium absorption is deficient and the balance between bone resorption and formation favors calcium uptake due to the high uncalcified osteoid surface. Typically, the serum phosphate level is also low, and hence, the product of calcium and phosphate is low. There is a raised parathyroid hormone concentration due to secondary hyperparathyroidism and,
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hence, an increased potential for phosphate excretion. These findings are the hallmark of classical osteomalaciaand rickets. It will also be appreciated that if the 1,25-dihydroxyvitaminD levels are low due to vitamin deficiency so are the levels of the precursor metabolites such as 25-hydroxyvitamin D. In fact, the adequacy of vitamin D nutrition is usually followed by measuring these precursors, typically 25-hydroxyvitamin D, as this is technically easier due to the about 100-fold higher levels. Further, in some cases 1,25-dihydroxyvitaminD levels may be near normal, depending on the phase of vitamin D depletion and the associated secondary hyperparathyroidism.
NOMENCLATURE OF VITAMIN D COMPOUNDS The term vitamin D is currently used for a number of closely related secosteroid compounds which have the potential to prevent rickets. Vitamin Dl, the original active substance isolated by Windaus, turned out to be a 1:1 molecular compound of lumisterol and vitamin D2; the former has the same molecular weight as vitamin D2 (396.63), differs from ergosterol only in that the methyl group at C19 is above the steroid ring and can be produced from ergosterol by ultraviolet irradiation. Vitamin D2 refers to 9,lO-secoergosta-5,7,10(19),22-tetra-en-, and has variously been referred to as calciferol, ergocalciferol, oleovitamin D2 and activated or cholecalciferol, ergosterol. Vitamin D3, 9,lO-secocholesta-5,7,10(19)-trien-3-01, has also been referred to as activated 7-dehydrocholesterol. Vitamin D4, 9,lO-secoergosta-5,7,10(19)-trien-3-01,is 22,23-dihydrovitamin D2 or 22,23-dihydroergocalciferol. 25-hydroxyvitamin D3 is also called calcifediol and 1-a, 25-dihydroxyvitamin D3 calcitriol; analogously, the drug 1-a-hydroxycholecalciferol is called alfacalcidol. Dihydrotachysterol is (5E,7E,22E)- 10-a-9, 10-secoergosta-5,7,22-trien-3-beta-01.
SOME FURTHER ASPECTS The various aspects of vitamin D dealt with below are meant to expand what has so far been presented and to provide material to illustrate the many actions of the vitamin in different situations, as well as focus on examples of the mode of action of the hormone and show how such an understanding provides insight into disease states. Renal Vitamin D Metabolism
The renal 1-a-vitamin D hydroxylase, stimulated by phosphate deficiency and parathyroid hormone (PTH) may also be induced by deficiency of vitamin D metabolites themselves. Growth hormone may also increase calcitriol levels, presumably by stimulating the renal la-hydroxylase. However, in diabetes melli-
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tus, calcitonin, the bone-protective hormone secreted in hypercalcemic states, may provide a stronger stimulus for I-a-hydroxylase induction than even PTH. In vitamin D deficiency the phosphaturic effects of injected PTH are lessened, although 25-OH-vitamin D diminishes the phosphaturic (and CAMP-uric)effects of PTH. Vitamin D deficiency is also associated with aminoaciduria which is not reversed by 1,25-dihydroxyvitaminD, and unrelated to increased PTH levels but may be mimicked by phosphate depletion. Though 1,25-dihydroxyvitaminD primarily acts to stimulate intestinal calcium and phosphate absorption and plays a role in resorption of bone (and possibly in its orderly mineralization), there is also evidence that it has an influence on the renal handling of calcium and phosphate. For example, 1,25-dihydroxyvitaminD may stimulate the renal ATP-dependent calcium pump.
Vitamin D and the Parathyroid Glands 1,25-Dihydroxyvitamin D acts on the parathyroid glands to suppress PTH secretion. However, if given clinically for this purpose to patients with renal failure to prevent secondary hyperparathyroidism, it may cause hypercalcemia due to its action on the intestine and bone. An analogue, 22-oxa-1,25(OH&D3,may be better for this purpose because it depresses hyperparathyroidism but does not cause hypercalcemia. Due to its conversion to 1,25-dihydroxyvitaminD, l-a-hydroxyvitamin D may also suppress hyperparathyroidism and per pg of dose may give rise to less hypercalcemia. In primary hyperparathyroidism, when the patient has sufficient vitamin D, there is hyperabsorption of calcium from the intestine; when the patient is vitamin D deficient, the prime action of increased PTH is on bone which becomes demineralized and fibrosed.
Vitamin D Deficiency and the Parathyroid Glands In secondary hyperparathyroidism which is a characteristic of vitamin D deficiency, there is phosphaturia, bicarbonaturia, and amino aciduria. Also sodium dependent uptake of amino acids at the apical membrane of the proximal renal tubule is decreased in vitamin D deficiency. The raised PTH in vitamin D deficiency is probably due to reduced parathyroid suppression caused by a low 1,25-dihydroxyvitamin D level, so that there is hyperplasia and an increase in the quantity of releasable hormone due to increased synthetic activity normally suppressed by PTH itself when vitamin D levels are normal.
Vitamin D in Relation to Certain Other Diseases X-Linked Hypophosphatemic Vitamin D Deficient Rickets
Raised intracellular phosphate due to defective phosphate handling by the kidneys suppresses the 1-a-hydroxylase enzyme leading to deficient production of
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1,25-dihydroxyvitamin D. Hence treatment with 1-a-hydroxyvitamin D will help the diminished calcium and phosphate absorption in this disease and also suppress parathyroid hyperplasia. This approach is preferable to vitamin D and phosphate supplementation as the latter may further depress renal 1-a-hydroxylation by raising phosphate levels. A somewhat similar situation prevails in the hyperphosphatemia of chronic renal failure where calcitriol is also low, with parathyroid hyperplasia. Vitamin D Resistant Rickets
There are two special forms of rickets which may respond only to very large doses of vitamin D: vitamin D resistant rickets types I and 11. Recent work has shown that in type I there is deficient activity of the 1-a-hydroxylase enzyme in the kidney proximal tubule, at least in some of the cases investigated. In type I1 there is ample activity of this enzyme but there is a target tissue receptor defect (or a postreceptor defect) for 1,25-dihydroxyvitamin D so that, for example, calcium transport from bone and absorption from the intestine are defective. Obviously, sufficient calcium supplementation is always necessary in type 11, to "overdrive" calcium absorption, which is partly vitamin D independent. Pseudohypoparathyroidism
In pseudohypoparathyroidism, where there is an apparent sufficiency of parathyroid hormone with raised serum levels, but a low serum calcium level and tissue resistance to PTH, only the urinary hydroxyprolinelcreatinine ratio (an indicator of bone collagen breakdown), though not osteocalcin or alkaline phosphatase (markers of bone tissue formation), is raised. This is diagnostic of a refractory bone response. Even when the serum calcium level is normalized by vitamin D or 1,25-dihydroxyvitamin D treatment, the serum PTH level remains raised, indicating a higher parathyroid gland set point for serum calcium in this disease. Newer serum and urinary collagen metabolites are increasingly used in assessing bone diseases.
SUMMARY Vitamin D was discovered following studies of the deforming bone disease known as rickets (osteomalacia in the adult). This vitamin is a secosteroid with an open B-ring. After 25-hydroxylation in the liver and 1-a hydroxylation in the kidney, the vitamin is an active hormone. It is a potent stimulator of calcium absorption from the intestine via binding of a phosphorylated receptor- 1,25-dihydroxyvitamin D complex to specific sites on DNA. This DNA binding in the intestine, bone, and other cells causes unfolding of DNA with subsequent transcription for calcium
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binding proteins, and other important proteins including osteocalcin and alkaline phosphatase. These inducing actions of the receptor-hormone complex are vital to health at the organ, tissue, and cellular level; differentiation of malignant cells can be induced by the complex. Because it is a potent hormone which, when in excess can cause potentially fatal hypercalcemia, it is regulated by many feedback systems. Parathyroid hormone and the low phosphate levels that it causes in renal proximal tubule cells induce renal 1-a-hydroxylase activity, and diminishing levels of PTH with higher phosphate levels reduce this enzyme's activity; hence, 1,25-dihydroxyvitamin D levels may be controlled up or down, respectively. The major serum metabolite of vitamin D is 25-hydroxyvitamin D which is carried in the serum by vitamin D binding protein and measurement of this metabolite's levels provide the best indicator of vitamin status, clinically. The main excretory form of hormonal vitamin D is calcitroic acid, a product of side-chain cleavage excreted mainly in bile. The simple vitamin D deficiency of classical rickets, either due to deficient dietary intake or diminished skin synthesis of vitamin D3, from 7-dehydrocholesterol or both, can be mimicked by diseases where vitamin D tissue receptors are deficient, where postreceptor mechanisms are disturbed, or by deficient production of hydroxylated forms due to enzyme abnormalities. Conversely, hypercalcemia may be caused by overactivity of vitamin D mechanisms, e.g., by vitamin D poisoning, or in diseases like sarcoidosis where there are cells excessively hydroxylating vitamin D to its hormonal form. Pharmacological forms of vitamin D with side-chain or steroid-ring alterations and substitutions are being sought for therapeutic use in controlling cell differentiation without causing hypercalcemia, e.g., calcipotriol for psoriasis.
RECOMMENDED READINGS Dabek, J. (1990). Anemerging view of vitarninD. Scand. J. Clin. Lab. Invest. 50,201, (Suppl.), 127-133. DeLuca, H.F., Krisinger, J., & Darwish, H. (1990). The vitamin D system: Kidney Int. 38, (Suppl. 29). S2-8. Eisman, J.A. (1988). Osteomalacia. In: Bailliere's Clinical Endocrinology and Metabolism (Martin, T.J., ed.), pp. 25-155. Balliere, London. Norman, A.W. (1992). Bone biochemistry and physiology from the perspectives of the vitamin D endocrine system. Curr. Op. Rheum. 4,375-382.
The Prehormone Vitamin D
JAN T. DABEK
History Rickets and Osteomalacia The Chemistry of Vitamin D Dietary Requirement of Vitamin D Vitamin D Metabolism The Vitamin D Endocrine System The Cellular Receptor for 1-a,25-DihydroxyvitaminD Transcription Induced by Active 1,25-DihydroxyvitaminD Receptor Vitamin D Transport Protein Vitamin D and Classical Rickets Nomenclature of Vitamin D Compounds Some Further Aspects Renal Vitamin D Metabolism Vitamin D and the Parathyroid Glands Vitamin D Deficiency and the Parathyroid GIands Vitamin D in Relation to Certain Other Diseases Summary
HISTORY That the childhood bone deforming disease rickets could be induced by deficiency of a fat soluble substance was shown in 1919by Mellanby. While Mellanby thought Principles of Medical Biology, Volume 8C Molecular and Cellular Pharmacology,Pages 933-949. Copyright O 1997 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-813-7
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that the factor was vitamin A, McCollum showed that a new substance was involved and gave it the name vitamin D. The situation was complicated by the finding by Huldshinsky that not only fish liver oil but also ultraviolet light could in some way provide the factor. Steenbock and Black showed that even ultraviolet irradiation of food could act to induce the factor. This led to the possibility of preparing sufficient amounts of vitamin D for the structure of vitamin D2 (the plant fat form) to be determined by Askew and Windaus in the early 1930s. As a consequence of this advance in chemistry, it was possible to synthesize another active form, vitamin D3 which Schenck identified as the form of vitamin D in animal fat. In the last 20 years or so, due to the availability of highly radiolabeled but relatively stable vitamin D, other forms of the vitamin have been discovered. These include two crucial hydroxylated forms named 25-hydroxyvitamin D and 1-a, 25-dihydroxyvitamin D. The former is the main metabolite of vitamin D produced by the liver, and the latter the hormonal form produced in the kidney which is involved in calcium absorption and bone remodeling.
RICKETS A N D OSTEOMALACIA The significance of vitamin D is inextricably bound up with the history and understanding of the bone diseases rickets, in children, and osteomalacia in adults. These diseases are primarily associated with disordered bone growth, decreased bone strength, and deformity resulting from inadequatecalcification of bone matrix in cartilaginous or fibrous osteogenesis. In children the long bones do not grow normally and the epiphyseal growth plate is thickened leading, in advanced cases, to palpable nodulation near the ends of the long bones. The platelike fibrous bones, such as those of the vault of the skull, can be indented by pressure, a sign called craniotabes, and the face takes on a rectangular form. The junctions between calcified rib and costal cartilage are nodularly expanded giving rise to a sign called the rachitic rosary. The legs become bowed and the pelvis deformed by weight bearing, which in girls may later result in difficulties in parturition. In the absence of treatment, the stature remains short and after the closing of the epiphyses the deformities remain. The gait tends to be waddling due to both muscular weakness and pelvic deformity; weakness of the abdominal muscles results in abdominal distension. In the adult there is bone pain and so-called pseudofractures on X-ray (Milkman's fractures, Looser's zones), which are small areas of increased radiolucency due to markedly lessened calcification in cartilaginous bones. These are perhaps most common in the rami of the pubis and the neck of the femur. Histologically the main finding is decreased calcification of newly formed bone matrix which appears as zones of clearly undercalcified tissue along bony trabeculae. In marginal cases of vitamin D deficiency, symptoms may show exacerbations in the darker seasons, during growth spurts in children (adolescence), and during pregnancy, where the needs of the fetus take priority over those of the mother. In
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addition to the above features, advanced cases of osteomalacia have been earlier thought to show inadequate hematopoiesisand poor resistance to infection, features which may have been given a new significance in the light of recent research into the ramifying effects of hydroxylated vitamin D metabolites on cellular differentiation.
THE CHEMISTRY OF VITAMIN D Vitamin Dg or cholecalciferol (C27H440) refers to the secosteroid (a steroid with an opened ring) which can well be thought of as deriving from the steroid 7-dehydrocholesterol (Figure 1) by opening of its r ring between its ninth and tenth carbon atoms. Subsequent turning of the A-ring through 180" around bond C6-C7 makes dangles below the C- and D-rings with C3 at the lower left extreme, linked to them by the "remains" of the B-ring (Figure 2). There is one hydroxy group at C3 and three conjugated double bonds atC5-C6, C7-C8, and C 10-C19, which gives vitamin D-compounds their strong absorption at 265 nm (molar extinction coefficient about 460 per mol/l per cm). Vitamin D2 only differs as far as the side-chain of its natural precursor, ergosterol, differs from that of 7-dehydrocholesterol, i.e., it has a double bond between C22-C23 and a methyl replacing one of the hydrogens at C24. There are also other forms of vitamin D, such as vitamin D4 and D5with
Figure 7.
The structure of 7-dehydrocholesterol.
JAN T. DABEK
Figure 2. The structure of vitamin D3.
slightly differing side-chains but these are not of essential physiological interest. Ergosterol and 7-dehydrocholesterol, the natural precursors of vitamin D in plants and animals, respectively, are found in nature more widely than the vitamin forms themselves which seem mainly to be formed by exposure of the former to ultraviolet light of suitable wavelength (about 280 to 310 nm) with subsequent thermal rearrangement. Other wavelengths may destroy vitamin D by converting it to other closely related but non-vitamin compounds.
The Prehormone Vitamin D
DIETARY REQUIREMENT OF VITAMIN D Each microgram of vitamin D corresponds to 40 international units of antirachitic activity. The normal daily requirement is 200 to 400 iu or 5 to 10 pg, with the higher level during growth and development and the lower in mature adults. This can be obtained from fortified milk (usually as vitamin D2), fish liver oils (vitamin D3), butter, egg yolk, liver, or from ultraviolet light acting on skin precursors. During pregnancy and lactation a supplement of an extra 5 pg per day has been recommended. For treatment of dietary or other forms of vitamin D deficiency-hypoparathyroidism and renal osteomalacia-thousands and tens of thousands of units of the vitamin may be required or then treatment is with hydroxylated forms of the vitamin according to the latest research findings.
VITAMIN D METABOLISM On entry to the extracellular fluid space or blood, vitamin D is rapidly bound to its transport protein, vitamin D-binding globulin (DBP), and then taken up within an hour or so by the liver. The liver has two enzyme systems for hydroxylation of the vitamin at carbon-25, to give 25-hydroxyvitamin D (Figure 3), a more active microsomal mono-oxygenase and a less active mitochrondrial enzyme that may be more relevant in states of vitamin D repletion or excess. The microsomal enzyme involves a specific NADPH-cytochrome P-450 reductase which seems to be inhibited by 1,25-dihydroxyvitamin D and low or high calcium levels. The 25-hydroxyvitamin D secreted by the liver, even within a few hours of initial vitamin D uptake, is hydroxylated in the 1-aposition (the a means that the hydroxy group is below the plane of the steroid rings as normally oriented in acyclopentanophenanthrene backbone) by a renal proximal tubule hydroxylase to give 1-a,25-dihydroxyvitamin D (Figure 4). It is located in the inner part of mitochondria and is a three-component mixed-function oxidase. The functioning of this enzyme involves successive reduction along the chain NADPH, renal ferridoxin reductase (a flavoprotein loosely named renal ferridoxin), then a cytochrome P-450. The enzyme is stimulated by parathyroid hormone, though perhaps more directly by the reduced phosphate levels this hormone brings about; the prevailing calcium level may also independently modulate the enzyme, and there is some evidence for a stimulatory role for growth hormone, prolactin and calcitonin. These are the two most important hydroxylations and 1,25-dihydroxyvitamin D is regarded as the main hormonal form of vitamin D. There are also, however, many other hydroxylated forms, such as 24,25-dihydroxyvitamin D, the renal hydroxylase induced by 1,25-dihydroxyvitamin D. This 24,25-dihydroxyvitamin is active on cartilage in some stages of growth and development and it may have some longer term actions on bone, but these are poorly defined and difficult to unravel. Other hydroxylated forms are 25,26-dihydroxyvitamin D and 1,24,25-trihydroxyvitamin D. There is also a 23,25-dihydroxy- and a 23,25,26-trihydroxyvitaminD; the latter
JAN T. DABEK
Figure 3.
The structure of 25-hydroxyvitamin D3.
may transform to the 25-hydroxy,26,23-lactoneform. The hydroxylation at the 24-position is in the R-orientation but in the 23-position in the S-orientation. (Viewed from the direction of the lightest side-group at a tetrahedral carbon atom the remaining groups are arranged in clockwise (R) or anticlockwise (S) order by increasing weight or group priority according to an agreed international scheme.) 1,25-dihydroxyvitamin D is metabolized to a 23-carboxylic acid called calcitroic acid, a polar inactived form, by target cell metabolism, possibly via 24R-
The Prehorrnone Vitamin D
Figure 4.
The structure of I-a,25-dihydroxyvitarnin D3.
then 23-hydroxylations prior to side-chain cleavage. Glucuronides are also formed and secreted in bile and urine; they may be hydrolyzed in the bowel so that enterohepatic circulation of vitamin D metabolites occurs. In addition to these main hydroxylations in the liver and kidney, the placenta can also 1-a hydroxylate vitamin D thus providing an additional reserve in pregnancy. The early observations ascribing to the kidney a unique role in 1-a hydroxylation of vitamin D have been modified, particularly since vitamin D
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metabolites have emerged as paracrine, even perhaps as autocrine, factors involved in tissue differentiation and response to challenges such as cancerous disease and infection. These tissue level hydroxylations do not, perhaps, normally influence gross systemic vitamin D metabolite levels but they are important local events which, in the case of exaggerated responses, could influence gross vitamin D metabolism. Macrophages in particular have been shown to be capable of 1-a hydroxylation of vitamin D and with massive macrophage infiltration systemic 1,25-dihydroxyvitamin D levels may be raised.
THE VITAMIN D ENDOCRINE SYSTEM The conversion of vitamin D to its active hormonal form by hydroxylation of 25-hydroxyvitamin D to 1-a, 25-dihydroxyvitamin D in the kidney is indirectly under the control of the serum calcium level (Figure 5). When the serum ionized calcium level falls, the increased secretion of parathyroid hormone from the parathyroid glands either directly stimulates proximal renal tubular cells to hydroxylate more 25-hydroxyvitamin D to 1,25-dihydroxyvitaminD or accomplishes this via a phosphate lowering effect. It will be recalled that parathyroid hormone stimulates renal phosphate excretion, clearly another factor in addition to increased release of calcium from bone that raises serum and tissue fluid ionized calcium levels. As more 1,25-dihydroxyvitamin D enters the circulation more is able to accumulate in the nuclei of intestinal target cells to stimulate calcium absorption from secreted fluids and chylous food. This raises ionized calcium levels even more and eventually reduces parathyroid hormone secretion, accomplishing the aim of normalizing the ionized calcium level. As the parathyroid stimulus to the renal tubule falls off, sodoes I-a hydroxylation of 25-hydroxyvitamin D with subsequent decay of serum levels with lessening of intestinal calcium absorption. The mechanism by which 1,25-dihydroxyvitaminD acts is not yet fully understood but it is certain that it binds to a receptor protein in target cells and the complex to DNA, where it acts as a transcription factor for relevant genes such as the calcium binding proteins (Figure 6). It also seems to stimulate the activation of other genes as, for example, in cell culture where cell differentiation can be demonstrated; this indicates coordination of genetic actions by either stimulation of many genes or by taking part in a series of processes that together coordinate the action of many genes. For example, hematopoietic cell lines can be caused to differentiate along the monocyte-macrophage axis. There are cotranscription factors involved in genomic control in addition to the 1,25-dihydroxyvitaminD-receptor complex which binds to this to modulate transcription activity. Such factors are the transcription regulatory proteins which also modulate vitamin A action by complexing with receptors. The genetic actions of vitamin D require a lag phase of about two-hours, but there are also some more immediate effects such as an early entry of calcium into the enterocyte which are thought to occur perhaps by cell membrane effects. The
Parathyroids
'
ip Liver ! Vitamin D-25 hydroxylase
PTH
4 \
25-OH-D-1 hydroxylase
Bone '
I[ Figure 5. Vitamin D, its hydroxylation and hormonal control of the calcium level. 941
Figure 6. The 1,25-dihydroxyvitamin D-receptor protein complex binds to chromosomal DNA to initiate chain unwinding and transcription. Other small modulator proteins may act as co-transcription factors at various genomic sites.
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Tabk 1. Levels of Vitamin D Involvement Organism
dietary input, skin synthesis, biliary excretion, blood calcium homeostasis
Organ
intestinal calcium absorption, skeletal calcification, calcium removal from skeleton
Tissue
hydroxylated in the liver and kidney (and placenta)
Cell
binds to a receptor protein which interacts with nuclear DNA to promote transcription; has a direct effect on the cell membrane
Table 2. Physiological Effects of Vitamin D increases intestinal calcium absorption Facilitates calcium resorption from bone Facilitates bone calcification Augments intestinal phosphate transport Facilitates renal handling of calcium and phosphate Has effects on cell differentiation Influences function of endocrine glands
levels of involvement of vitamin Dare summarized in Table 1, and the major effects of 1,25-dihydroxyvitaminD are listed for convenience in Table 2.
THE CELLULAR RECEPTOR FOR 1-a, 25-DIHYDROXWITAMIN D The nuclear receptor for 1,25-dihydroxyvitamin D is a 55 kD protein located widely at low concentration in diverse cells. It is, of course, predominantly present in the major endocrine target tissues-intestine, kidney, bone-but also in the parathyroid glands, the islet cells of the pancreas, the mammary gland and many other sites as is seen in Table 3. In addition, vitamin D receptors are found in osteoclast precursors (monocytes) and also at notable levels in many cancer cells. Increased expression of the receptor for 1,25-dihydroxyvitamin D, termed up-regulation, is in part dependent on the levels of 1,25-dihydroxyvitaminD itself and up-regulation may be followed by suppression. The receptor can also be induced by glucocorticoids,which may be an important event in the neonatal period when initiation of intestinal absorption of dietary calcium is, upon complete termination of placental calcium transfer, an immediate necessity of postnatal life. The receptor has binding regions for 1,25-dihydroxyvitaminD, a region with a molecular mass of about 23,000 Da, and separated from this by a hingelike region
JAN T. DABEK
Table 3. Tissues With Vitamin D Receptors Intestinal mucosa Kidney tubule Cells of bone tissue Parathyroid Islets of Langerhans Mammary gland Keratinocytes Fibroblasts
Epithelial cells Thymocytes Lymphocytes Hematopoietic precursor cells Sertoli cells Ovarian cells Heart cells
Table 4 . The Steroid Receptor Family I. The vitamin D receptor is alike in different species. II. The following receptors are all transcription factors and similar in structure and mode of action. Vitamin D receptor Glucocorticoid receptor Retinoic acid receptor Thyroid hormone receptor Estrogen receptor
is a smaller DNA-binding stretch towards the amino end. The structure of the receptor is similar in different species. Other steroid receptors (estrogen, glucocorticoid), the thyroid hormone receptor, and a retinoic acid receptor have a clearly similar structure and these all act as gene transcription factors, as listed in Table 4. The vitamin D receptor may require phosphorylation at certain sites before it is fully competent as a transcription factor; it shows genetic polymorphism which could be related to disease patterns, e.g., osteoporosis.
TRANSCRIPTION INDUCED BY ACTIVE 1,25-DIHYDROWITAMIN D RECEPTOR Calcium binding proteins, such as calbindin-28 and calbindin-9, are induced in target cells by 1,25-dihydroxyvitamin D. These are involved in calcium transport, for example, across the bowel mucosa. Though the whole mechanism of calcium transport within cells and across cellular membranes cannot be explained by calcium binding proteins alone, the genes for these proteins have been studied and 5'-flanking promoter regions with specific palindromic nucleotide sequences responsive to the 1,25-dihydroxyvitamin D-receptor complex have been identified. Other proteins, like osteocalcin and alkaline phosphatase are also induced by hormonal vitamin D in target tissue (Table 5). This does not mean that only hormonal vitamin D elicits these responses; the same genes may be controlled by a number of factors. For example, the calcium binding protein of the uterus may be induced by estrogens.
The Prehormone Vitamin D Table 5 . Proteins Induced or Regulated by 1,25-Dihydroxyvitamin D via its Nuclear Receptor Calbindin-28 Calbindin-9 Alkaline phosphatase Osteocalcin
Ornithine decarboxylase ca2+-~~pase Renal cyclic-AMP-dependent protein kinase inhibitor
VITAMIN D TRANSPORT PROTEIN The serum DBP transports not only the vitamin in the serum but also its metabolites. It is also called vitamin D transport protein or group specific component (Gc) of which there are at least three slightly differing genetic forms all with similar vitamin binding constants. It is an albuminlike protein with an association equilibrium constant of about 5 x 10' for vitamin D. For the metabolites the associationconstants are 1 x 10' for 24,25-dihydroxyvitaminD and 1 x lo6 for 1,25-dihydroxyvitamin D. It prevents unnecessary loss of secosteroid via renal filtration and maintains a reservoir of the various metabolites ready for metabolic needs. The clearly lower affinity of this protein for 1,25-dihydroxyvitamin D facilitates the more rapid dissociation of the main hormonal form of the vitamin for speedy endocrinological effect. The level of the protein in blood plasma is about 350 mgll. In pregnancy this level may increase and thereby buffer the free concentrations of increased total serum 1,25-dihydroxyvitamin D hormone downwards. The molecular weight of DBP, which is secreted by the liver, is about 54-56,000 and the molecule contains about 6% carbohydrate. Recent DNA-level research has revealed the genes for vitamin D binding protein and the 1,25-dihydroxyvitaminD receptor protein. A virtually full-length cDNA for human serum vitamin D transport protein has been obtained from human liver messenger RNA using an expression library. Complete sequencingyielded the code for 458 amino acid residues with a calculated molecular weight of 51,335. The amino acid sequence shows strong homology with human serum albumin and a-fetoprotein.
VITAMIN D AND CLASSICAL RICKETS Without adequate serum levels of 1,25-dihydroxyvitaminD it is not possible for homeostatic mechanisms to maintain the serum calcium level (about 2.5 rnmolA) relatively constant. This is because intestinal calcium absorption is deficient and the balance between bone resorption and formation favors calcium uptake due to the high uncalcified osteoid surface. Typically, the serum phosphate level is also low, and hence, the product of calcium and phosphate is low. There is a raised parathyroid hormone concentration due to secondary hyperparathyroidism and,
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hence, an increased potential for phosphate excretion. These findings are the hallmark of classical osteomalaciaand rickets. It will also be appreciated that if the 1,25-dihydroxyvitaminD levels are low due to vitamin deficiency so are the levels of the precursor metabolites such as 25-hydroxyvitamin D. In fact, the adequacy of vitamin D nutrition is usually followed by measuring these precursors, typically 25-hydroxyvitamin D, as this is technically easier due to the about 100-fold higher levels. Further, in some cases 1,25-dihydroxyvitaminD levels may be near normal, depending on the phase of vitamin D depletion and the associated secondary hyperparathyroidism.
NOMENCLATURE OF VITAMIN D COMPOUNDS The term vitamin D is currently used for a number of closely related secosteroid compounds which have the potential to prevent rickets. Vitamin Dl, the original active substance isolated by Windaus, turned out to be a 1:1 molecular compound of lumisterol and vitamin D2; the former has the same molecular weight as vitamin D2 (396.63), differs from ergosterol only in that the methyl group at C19 is above the steroid ring and can be produced from ergosterol by ultraviolet irradiation. Vitamin D2 refers to 9,lO-secoergosta-5,7,10(19),22-tetra-en-, and has variously been referred to as calciferol, ergocalciferol, oleovitamin D2 and activated or cholecalciferol, ergosterol. Vitamin D3, 9,lO-secocholesta-5,7,10(19)-trien-3-01, has also been referred to as activated 7-dehydrocholesterol. Vitamin D4, 9,lO-secoergosta-5,7,10(19)-trien-3-01,is 22,23-dihydrovitamin D2 or 22,23-dihydroergocalciferol. 25-hydroxyvitamin D3 is also called calcifediol and 1-a, 25-dihydroxyvitamin D3 calcitriol; analogously, the drug 1-a-hydroxycholecalciferol is called alfacalcidol. Dihydrotachysterol is (5E,7E,22E)- 10-a-9, 10-secoergosta-5,7,22-trien-3-beta-01.
SOME FURTHER ASPECTS The various aspects of vitamin D dealt with below are meant to expand what has so far been presented and to provide material to illustrate the many actions of the vitamin in different situations, as well as focus on examples of the mode of action of the hormone and show how such an understanding provides insight into disease states. Renal Vitamin D Metabolism
The renal 1-a-vitamin D hydroxylase, stimulated by phosphate deficiency and parathyroid hormone (PTH) may also be induced by deficiency of vitamin D metabolites themselves. Growth hormone may also increase calcitriol levels, presumably by stimulating the renal la-hydroxylase. However, in diabetes melli-
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tus, calcitonin, the bone-protective hormone secreted in hypercalcemic states, may provide a stronger stimulus for I-a-hydroxylase induction than even PTH. In vitamin D deficiency the phosphaturic effects of injected PTH are lessened, although 25-OH-vitamin D diminishes the phosphaturic (and CAMP-uric)effects of PTH. Vitamin D deficiency is also associated with aminoaciduria which is not reversed by 1,25-dihydroxyvitaminD, and unrelated to increased PTH levels but may be mimicked by phosphate depletion. Though 1,25-dihydroxyvitaminD primarily acts to stimulate intestinal calcium and phosphate absorption and plays a role in resorption of bone (and possibly in its orderly mineralization), there is also evidence that it has an influence on the renal handling of calcium and phosphate. For example, 1,25-dihydroxyvitaminD may stimulate the renal ATP-dependent calcium pump.
Vitamin D and the Parathyroid Glands 1,25-Dihydroxyvitamin D acts on the parathyroid glands to suppress PTH secretion. However, if given clinically for this purpose to patients with renal failure to prevent secondary hyperparathyroidism, it may cause hypercalcemia due to its action on the intestine and bone. An analogue, 22-oxa-1,25(OH&D3,may be better for this purpose because it depresses hyperparathyroidism but does not cause hypercalcemia. Due to its conversion to 1,25-dihydroxyvitaminD, l-a-hydroxyvitamin D may also suppress hyperparathyroidism and per pg of dose may give rise to less hypercalcemia. In primary hyperparathyroidism, when the patient has sufficient vitamin D, there is hyperabsorption of calcium from the intestine; when the patient is vitamin D deficient, the prime action of increased PTH is on bone which becomes demineralized and fibrosed.
Vitamin D Deficiency and the Parathyroid Glands In secondary hyperparathyroidism which is a characteristic of vitamin D deficiency, there is phosphaturia, bicarbonaturia, and amino aciduria. Also sodium dependent uptake of amino acids at the apical membrane of the proximal renal tubule is decreased in vitamin D deficiency. The raised PTH in vitamin D deficiency is probably due to reduced parathyroid suppression caused by a low 1,25-dihydroxyvitamin D level, so that there is hyperplasia and an increase in the quantity of releasable hormone due to increased synthetic activity normally suppressed by PTH itself when vitamin D levels are normal.
Vitamin D in Relation to Certain Other Diseases X-Linked Hypophosphatemic Vitamin D Deficient Rickets
Raised intracellular phosphate due to defective phosphate handling by the kidneys suppresses the 1-a-hydroxylase enzyme leading to deficient production of
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1,25-dihydroxyvitamin D. Hence treatment with 1-a-hydroxyvitamin D will help the diminished calcium and phosphate absorption in this disease and also suppress parathyroid hyperplasia. This approach is preferable to vitamin D and phosphate supplementation as the latter may further depress renal 1-a-hydroxylation by raising phosphate levels. A somewhat similar situation prevails in the hyperphosphatemia of chronic renal failure where calcitriol is also low, with parathyroid hyperplasia. Vitamin D Resistant Rickets
There are two special forms of rickets which may respond only to very large doses of vitamin D: vitamin D resistant rickets types I and 11. Recent work has shown that in type I there is deficient activity of the 1-a-hydroxylase enzyme in the kidney proximal tubule, at least in some of the cases investigated. In type I1 there is ample activity of this enzyme but there is a target tissue receptor defect (or a postreceptor defect) for 1,25-dihydroxyvitamin D so that, for example, calcium transport from bone and absorption from the intestine are defective. Obviously, sufficient calcium supplementation is always necessary in type 11, to "overdrive" calcium absorption, which is partly vitamin D independent. Pseudohypoparathyroidism
In pseudohypoparathyroidism, where there is an apparent sufficiency of parathyroid hormone with raised serum levels, but a low serum calcium level and tissue resistance to PTH, only the urinary hydroxyprolinelcreatinine ratio (an indicator of bone collagen breakdown), though not osteocalcin or alkaline phosphatase (markers of bone tissue formation), is raised. This is diagnostic of a refractory bone response. Even when the serum calcium level is normalized by vitamin D or 1,25-dihydroxyvitamin D treatment, the serum PTH level remains raised, indicating a higher parathyroid gland set point for serum calcium in this disease. Newer serum and urinary collagen metabolites are increasingly used in assessing bone diseases.
SUMMARY Vitamin D was discovered following studies of the deforming bone disease known as rickets (osteomalacia in the adult). This vitamin is a secosteroid with an open B-ring. After 25-hydroxylation in the liver and 1-a hydroxylation in the kidney, the vitamin is an active hormone. It is a potent stimulator of calcium absorption from the intestine via binding of a phosphorylated receptor- 1,25-dihydroxyvitamin D complex to specific sites on DNA. This DNA binding in the intestine, bone, and other cells causes unfolding of DNA with subsequent transcription for calcium
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binding proteins, and other important proteins including osteocalcin and alkaline phosphatase. These inducing actions of the receptor-hormone complex are vital to health at the organ, tissue, and cellular level; differentiation of malignant cells can be induced by the complex. Because it is a potent hormone which, when in excess can cause potentially fatal hypercalcemia, it is regulated by many feedback systems. Parathyroid hormone and the low phosphate levels that it causes in renal proximal tubule cells induce renal 1-a-hydroxylase activity, and diminishing levels of PTH with higher phosphate levels reduce this enzyme's activity; hence, 1,25-dihydroxyvitamin D levels may be controlled up or down, respectively. The major serum metabolite of vitamin D is 25-hydroxyvitamin D which is carried in the serum by vitamin D binding protein and measurement of this metabolite's levels provide the best indicator of vitamin status, clinically. The main excretory form of hormonal vitamin D is calcitroic acid, a product of side-chain cleavage excreted mainly in bile. The simple vitamin D deficiency of classical rickets, either due to deficient dietary intake or diminished skin synthesis of vitamin D3, from 7-dehydrocholesterol or both, can be mimicked by diseases where vitamin D tissue receptors are deficient, where postreceptor mechanisms are disturbed, or by deficient production of hydroxylated forms due to enzyme abnormalities. Conversely, hypercalcemia may be caused by overactivity of vitamin D mechanisms, e.g., by vitamin D poisoning, or in diseases like sarcoidosis where there are cells excessively hydroxylating vitamin D to its hormonal form. Pharmacological forms of vitamin D with side-chain or steroid-ring alterations and substitutions are being sought for therapeutic use in controlling cell differentiation without causing hypercalcemia, e.g., calcipotriol for psoriasis.
RECOMMENDED READINGS Dabek, J. (1990). Anemerging view of vitarninD. Scand. J. Clin. Lab. Invest. 50,201, (Suppl.), 127-133. DeLuca, H.F., Krisinger, J., & Darwish, H. (1990). The vitamin D system: Kidney Int. 38, (Suppl. 29). S2-8. Eisman, J.A. (1988). Osteomalacia. In: Bailliere's Clinical Endocrinology and Metabolism (Martin, T.J., ed.), pp. 25-155. Balliere, London. Norman, A.W. (1992). Bone biochemistry and physiology from the perspectives of the vitamin D endocrine system. Curr. Op. Rheum. 4,375-382.