Synthesis of (6-3H)-1alpha-hydroxyvitamin D3 and its metabolism in vivo to (3H)-1alpha,25-dihydroxyvitamin D3.

[6-3H]-1alpha-Hydroxyvitamin D3 was chemically synthesized and its full biological activity and radiochemical purity were demonstrated. With the use of this preparation it has been possible to demonstrate in vivo that in rats the [6-3H]-1Alpha-hydroxyvitamin D3 is converted to [6-3H]-1alpha,25-dihydroxyvitamin D3, the natural hormone. In fact, in the intestine and bone of rats given 32 picomoles of [6-3H]-1alpha-hydroxyvitamin D3 each day for 6 days, more than 80 percent of the lipid-soluble radioactivity exists as [6-3H]-1alpha,25-dihydroxyvitamin D3, a finding that suggests that much of the biological effectiveness of 1alpha-hydroxyvitamin D3 is due to its conversion to 1alpha,25-dihydroxyvitamin D3.

Photosynthesis of previtamin D3 in human skin and the physiologic consequences.

Photosynthesis of previtamin D3 can occur throughout the epidermis in the dermis when hypopigmented Caucasian skin is exposed to solar ultraviolet radiation. Once previtamin D3 is formed in the skin, it undergoes a temperature-dependent thermal isomerization that takes at least 3 days to complete. The vitamin D-binding protein preferentially translocates the thermal product, vitamin D3, into the circulation. These processes suggest a unique mechanism for the synthesis, storage, and slow, steady release of vitamin D3 from the skin into the circulation.

1 alpha,25-dihydroxyvitamin D3 induces maturation of the human monocyte cell line U937, and, in association with a factor from human T lymphocytes, augments production of the monokine, mononuclear cell factor.

The monocyte factor, interleukin 1, or other factors homologous with interleukin 1, modulates functions of a variety of cells, including T and B lymphocytes, synovial cells, and chondrocytes. We have reported that a human monocyte cell line, U937, produces interleukin 1 when incubated with a soluble factor from lectin-stimulated T lymphocytes. We have also shown that U937 cells have a specific cytosolic receptor for 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25[OH]2D3). We now report that 1 alpha,25(OH)2D3(10(-11)-10(-10) M) induces maturational changes in the U937 cells similar to those produced by conditioned medium from lectin-stimulated T lymphocytes (increase in Fc receptors and OKM1 binding and decrease in proliferation), but does not induce monokine production as measured by mononuclear cell factor activity. 1 alpha,25(OH)2D3 is 200-300-fold more effective than 25-hydroxyvitamin D3, which is consistent with the known biological potency of these vitamin D3 metabolites. 1 alpha,25(OH)2D3 and the lymphokine together markedly augment maturational effects and, in addition, augment monokine production. The specificity of the interaction is further demonstrated by the lack of augmentation of monokine production with 1 beta,25-dihydroxyvitamin D3 in the presence of lymphokine. These interactions of a classical hormone and the hormonelike product(s) of the immune system with U937 cells serve as a model for human monocyte/macrophage differentiation and suggest a role for these interactions in some aspects of inflammation.

A simple method for generation of antibodies with specificity for 1,25-dihydroxyergocalciferol and 1,25-dihydroxycholecalciferol.

A simple method for production of antisera with high affinity and selectivity for 1 alpha, 25-dihydroxyergocalciferol and 1 alpha, 25-dihydroxychole-calciferol is described. 1 alpha-Hydroxy-25,26,27-trisnorcholecalciferol-24-oic acid was coupled directly to bovine serum albumin. Rabbits immunized with this conjugate rapidly produced antibodies that bound 3H-1 alpha,-25-dihydroxycholecalciferol with high affinity and demonstrated nearly equal reactivity with 1 alpha, 25-dihydroxyergocalciferol and poor reactivity with 25-hydroxycholecalciferol; 24,25-dihydroxycholecalciferol; 25,26-dihydroxycholecalciferol; and 1 beta,25-dihydroxycholecalciferol. The use of one of these antisera has led to the development of a specific assay for 1 alpha,25-dihydroxyergocalciferol and 1 alpha,25-dihydroxycholecalciferol in human serum.

24-Dehydrovitamin D is potent inhibitor of rat liver microsomal vitamin D-25-hydroxylase.

A pathway has been described in the skin for the synthesis of 24-dehydrovitamin D3 (delta 24D3) from 24-dehydroprovitamin D3. The physiologic function of delta 24D3 is unknown, but has been proposed as a potential inhibitor of hepatic vitamin D-25-hydroxylase. We validated an assay for vitamin D-25-hydroxylase in rat hepatic microsomes, using nanomolar amounts of [3H]D3 as substrate, and found that delta 24D3 competitively inhibits vitamin D-25-hydroxylase activity. The apparent Ki was approximately 17 nM, indistinguishable from the Km of approximately 15 nM, suggesting that both delta 24D3 and cholecalciferol have similar affinity for the enzyme. We found no [3H]delta 24D3 in serum or liver extracts after repletion of vitamin D-depleted rats with [3H]vitamin D3 for 4 h or 6 days. A dose of 1 microgram delta 24D3 to vitamin D- and calcium-depleted rats was unable to promote any elevation in the 45Ca transport by everted duodenal sacs or to increase levels of plasma calcium: thus no evidence for biological conversion of delta 24D3 to vitamin D3 was observed. Further studies are needed to determine whether delta 24D3 is released from the skin to the circulation and is taken up by the liver, before physiological relevance can be attributed to this inhibitor.

Evaluation of a photolabile derivative of 1,25-dihydroxyvitamin D3 as a photoaffinity probe for 1,25-dihydroxyvitamin-D3 receptor in chick intestinal cytosol.

We evaluated the viability of 1 alpha, 25-dihydroxyvitamin D3-3 beta-[N-(4-azido-2-nitrophenyl)glycinate] (1,25-(OH)2-D3-ANG), an analog of 1 alpha, 25-dihydroxyvitamin D3 (1,25-(OH)2-D3) as a photoaffinity probe for 1,25-(OH)2-D3 receptor in chick intestinal cytosol. A competitive-binding assay revealed that chick intestinal cytosolic 1,25-(OH)2- D3 receptor bound to 1,25-(OH)2-D3-ANG approximately 20-times less effectively than it did to 1,25-(OH)2-D3. Irradiation of 1,25-(OH)2-D3- ANG in the presence of chick intestinal cytosolic preparation significantly diminished subsequent binding to 3H-1,25-(OH)2-D3, suggesting that the photoaffinity analog was covalently attached to the receptor. Therefore the nitroarylazide derivative of 1,25-(OH)2-D3 may be a valuable photoaffinity probe for the characterization of the 1,25-(OH)2-D3 receptor.

Isolation and identification of 24-dehydroprovitamin D3 and its photolysis to 24-dehydroprevitamin D3 in mammalian skin.

Until now it had been assumed that mammalian skin contains only one provitamin D, 7-dehydrocholesterol, that is eventually converted to vitamin D3 after the skin is exposed to sunlight. Examination by reverse phase high performance liquid chromatography of lipid extracts from young rat skin, however, led to the observation that 7-dehydrocholesterol is not the only provitamin D in rat skin. Another provitamin D, accounting for 22 +/- 3% of the total provitamin content of the skin, was resolved from 7-dehydrocholesterol, and, on the basis of ultraviolet spectrophotometry, mass spectrometry, and nuclear magnetic resonance spectrometry, was identified as 24-dehydroprovitamin D3 (cholesta-5,7,24-trien-3 beta-ol). This new cutaneous provitamin D is not unique to the rat because it was also detected in the skin of reptiles, amphibians, birds, aquatic mammals, and humans. To be certain that the cutaneous 24-dehydroprovitamin D3 was as susceptible as 7-dehydrocholesterol to ultraviolet photolysis, rat skin was exposed to ultraviolet radiation. A reverse phase high performance liquid chromatographic analysis of a lipid extract of rat skin previously exposed to ultraviolet radiation demonstrated the presence of both previtamin D3 and 24-dehydroprevitamin D3. Therefore, these observations demonstrate for the first time that mammalian skin has the capacity to produce not one but at least two different vitamin Ds.

Photoaffinity labeling of the rat plasma vitamin D binding protein with [26,27-3H]-25-hydroxyvitamin D3 3 beta-[N-(4-azido-2-nitrophenyl)glycinate].

It is well recognized that the vitamin D binding protein (DBP) is important for the transport of vitamin D, 25-hydroxyvitamin D (25-OH-D), and its metabolites. In an attempt to better understand the molecular-binding properties of this ubiquitous protein, we designed and synthesized a photoaffinity analogue of 25-OH-D3 and its radiolabeled counterpart. This analogue, 25-hydroxyvitamin D3 3 beta-[N-(4-azido-2-nitrophenyl)glycinate] (25-OH-D3-ANG), was recognized by the rat DBP and was about 10 times less active than 25-OH-D3 in terms of binding. Incubation of [3H]25-OH-D3 or [3H]25-OH-D3-ANG with rat DBP revealed that both compounds were specifically bound to a protein with a sedimentation coefficient of 4.1 S. Each was displaced with a 500-fold excess of 25-OH-D3. When [3H]25-OH-D3-ANG was exposed to UV radiation in the presence of rat DBP followed by the addition of a 500-fold excess of 25-OH-D3, there was no displacement of tritium from the 4.1S peak. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiographic analysis of [3H]25-OH-D3-ANG exposed to UV radiation in the presence of rat DBP followed by the addition of a 500-fold excess of 25-OH-D3 revealed one major band with a molecular weight of 52 000. These data provide strong evidence that [3H]25-OH-D3-ANG was covalently linked to the rat DBP. This photoaffinity probe should provide a valuable tool for the analysis of the binding site on this transport protein.

Synthesis of 1alpha-hydroxy [6-3H]vitamin D3 and its metabolism to 1alpha, 25-dihydroxy [6-3H]vitamin D3 in the rat.

1alpha-Hydroxy [6-3H]vitamin D3 has been synthesized with a specific activity of 4 Ci/mmol, and its metabolism in rats has been studied. It is rapidly converted to 1alpha,25-dihydroxy [6-3H]vitamin D3 in vivo. Following an intravenous or oral dose, a maximal concentration of 1alpha,25-dihydroxy [6-3H]vitamin D3 is found 2 and 4 hours, respectively, before the maximal intestinal calcium transport response is observed. Similarly, 1alpha,25-dihydroxy[6-3H]vitamin D3 accumulation in bone precedes the bone calcium mobilization response. It appears, therefore, that the biological activity of 1alpha-hydroxyvitamin D3 is largely, if not exclusively, due to its conversion to 1alpha,25-dihydroxy[6-3H]vitamin D3 1alpha-Hydroxy[6-3H]vitamin D3 and 1alpha,25-dihydroxy[6-3H]vitamin D3 appear in intestine equally well after an oral or an intravenous dose of 1alpha-hydroxy[6-3H]vitamin D3. However, much less of both 1alpha-hydroxy[6-3H]vitamin D3 and 1alpha,25-dihydroxy[6-3H]vitamin D3 appears in bone and blood after an oral than after an intravenous dose. A much reduced bone calcium mobilization response is also noted following an oral dose as compared to an intravenous dose of 1alpha-hydroxyvitamin D3, suggesting that oral 1alpha-hydroxyvitamin D3 is not utilized as well as intravenously administered material.

Metabolism of 1alpha-hydroxyvitamin D3 in the chick.

Chicks convert both orally and intravenously administered 1alpha-hydroxy[6-3H]vitamin D3 rapidly to 1alpha,25-dihydroxy[6-3H]vitamin D3. The maximal accumulation of 1alpha,25-dihydroxy[6-3H]vitamin D3 in intestine precedes the intestinal absorption response to 1alpha-hydroxyvitamin D3 by at least 2 hours. Oral administration results in the highest concentrations of 1alpha,25-dihydroxy[6-3H]vitamin D3 in intestine, giving a level about 1.5 times that achieved with an intravenous dose. On the other hand, an oral dose of 1alpha-hydroxy[6-3H]vitaminD3 gives much lower amounts of both 1alpha-hydroxy[6-3H]vitamin D3 and 1alpha,25-dihydroxy[6-3H]vitamin D3 in bone and blood than an intravenous dose, which suggests that the 1alpha-hydroxy[6-3H]vitamin D3 may not be utilized as well by the oral route as by an intravenous route. Liver homogenates from both rat and chick convert 1alpha-hydroxy[6-3H]vitamin D3 to 1alpha,25-dihydroxy[6-3H]vitamin D3. However, intestinal homogenates from chick, but not rat, can also cary out this conversion, which may account for the higher concentration of 1alpha,25-dihydroxy[6-3H]vitamin D3 found in the intestine of chicks given an oral dose of 1alpha-hydroxy[6-3H]vitamin D3.

Synthesis of [3 beta-3H]-3-epivitamin D3 and its metabolism in the rat.

3-Epivitamin D3, the 3 alpha epimer of vitamin D3, was synthesized, and its biological activity in the rat was evaluated. It was found to be approximately 4 times less active on a weight basis than vitamin D3 with respect to intestinal calcium transport, bone calcium mobilization, and calcification score as determined by the line-test assay. Tritiated 3-epivitamin D3 was prepared, and its metabolism in the rat was compared with that of vitamin D3 to investigate the reasons for this diminished activity. 3-Epivitamin D3 was converted to two polar metabolites, for which the chromatographic properties and the origin of biosynthesis (in the liver and kidney, respectively) correspond to 25-hydroxy-3-epivitamin D3 and 1 alpha,25-dihydroxy-3-epivitamin D3. The fact that the concentration of 1 alpha,25-dihydroxy-3-epivitamin D3 in the intestine is half that of 1 alpha,25-dihydroxyvitamin D3 may be one explanation for the reduced biological activity of this epimer.

The photoproduction of 1 alpha,25-dihydroxyvitamin D3 in skin: an approach to the therapy of vitamin-D-resistant syndromes.

Cutaneous 7-dehydrocholesterol, exposed to ultraviolet radiation, converts to previtamin D3, which in turn converts in skin to vitamin D3 and is carried into the circulation. We investigated the feasibility of the photochemical conversion in skin of hydroxylated derivatives of 7-dehydrocholesterol - such as 1 alpha, 25-dihydroxy-7-dehydrocholesterol (1 alpha, 25-(OH)2-7-DHC) - to the corresponding hydroxylated previtamin as an alternative method of delivery of 1 alpha, 25-dihydroxyvitamin D3 (1 alpha, 25-(OH)2-D3) to subjects who are deficient in the endogenous metabolite. In human volunteers and in vitamin-D-deficient rats [24-3H] 1 alpha, 25-(OH)2-D3 appeared in blood after [24-3H] 1 alpha, 25-(OH)2-7-DHC was applied to the skin and exposed to ultraviolet radiation. In anephric rats, intestinal calcium absorption and serum calcium levels were elevated after a topical dose of 1 alpha, 25-(OH)2-7-DHC combined with ultraviolet phototherapy. Delivery of equivalent doses of 1 alpha, 25-(OH)2-D3 through the skin and orally showed that there was more prolonged stimulation in intestinal calcium absorption and serum calcium elevation after cutaneous administration. The photochemical conversion of precursors may be useful in the treatment of patients with impaired vitamin-D metabolism.

Isolation and identification of previtamin D3 from the skin of rats exposed to ultraviolet irradiation.

The process of the photolytic activation of vitamin D precursor(s) in the skin has been elucidated by a detailed analysis of the products formed after ultraviolet light exposure. The photolytic product isolated from the skin of rats exposed to ultraviolet irradiation was identified as previtamin D3 by several criteria including its (a) characteristic ultraviolet absorption spectrum, (b) mass spectrum, and (c) thermal isomerization to vitamin D3, which itself was identified also by mass spectroscopy. Vitamin D3 per se was not formed by ultraviolet irradiation--vitamin D3 arises exclusively from the thermal conversion of previtamin D3. Detectable amounts of lumisterol3 or tachysterol3 were not seen.