Synthesis and bioassay of 3-deoxy-1α-hydroxyvitamin D3, an active analog of 1α,25-dihydroxyvitamin D3

Two synthetic routes to 3-deoxy-1α-hydroxyvitamin D₃, an analog of 1α,25-dihydroxyvitamin D₃, are described. One involved the six-step conversion of 1α,2α-epoxy-6,6-ethylenedioxy-5α-cholestan-3- one to 1α-acetoxycholest-5-ene, whereas, in the second, the same intermediate was prepared from 1α-hydroxycholesterol. Conversion of the Δ⁵-sterol to the required 5,7-diene was accomplished most efficiently via 7-keto and 7-tosylhydrazone intermediates. Bioassay of 3-deoxy-1α-hydroxyvitamin D₃ in the rat establishes that the analog can fulfill all common vitamin D functions including stimulation of intestinal calcium transport, mobilization of calcium and phosphate from bone, stimulation of growth, and calcification of bone. Direct comparison indicates the compound to have $\text{1}\text{20}$ to $\text{1}\text{50}$ of the activity of 1α-hydroxyvitamin D₃, but it acts with a time course indistinguishable from the latter.     

The site of 1α,25-dihydroxyvitamin D3 production in pregnancy

Metabolism of 25-hydroxyvitamin D₃ (25-OH-D₃) in pregnancy was investigated $\text{in}\text{vitro}$ in New Zealand White rabbits fed a rabbit chow. Kidney homogenates from pregnant mothers and fetuses were separately incubated with [³H]-25-OH-D₃. The homogenates from fetuses produced significant amounts of $\text{[}{}^3\text{H]-1α,25-dihydroxyvitamin}$ D₃ [1α,25-(OH)₂-D₃] from its precursor, while those from mothers predominantly produced [³H]-24,25-dihydroxyvitamin D₃ [24,25-(OH)₂-D₃]. The identity of the radioactive metabolites produced from [³H]-25-OH-D₃ was established by periodate cleavage and comigration with synthetic 1α,25-(OH)₂-D₃ or 24,25-(OH)₂-D₃ on high pressure liquid chromatography. These results clearly indicate that the fetal kidney is at least one of the sites of 1α,25-(OH)₂-D₃ synthesis in pregnancy.     

Studies on vitamin D metabolism: Catabolism of 1α, 25-dihydroxyvitamin D3 and 1α-hydroxyvitamin D3 to a metabolite inactive on bone mineral mobilization

A heretofore unknown metabolite of vitamin D₃ was isolated from the 1α,24,25-trihydroxyvitamln D₃ fraction of lipid extracts obtained from plasma of rats which were given intravenous or oral doses of 100 pmol/100 g of either 1α-hydroxyvitamin D₃ or 1α, 25-dihydroxy-vitamin D₃. Doses of 25–250 pmoles of the new metabolite when given to a vitamin D deficient rat were completely inactive in terms of stimulating the classic vitamin D response of bone calcium mobilization. The nature of the metabolism of 1α-hydroxyvitamin D₃ or 1α, 25-dihydroxy-vitamin D₃ to the metabolite is not clear at the present time, but it is probable that neither of these steroids undergo side-chain cleavage to yield the new metabolite.     

Chemical synthesis of [1β-3H]1α,25-dihydroxyvitamin D3 and [1α-3H]1β,25-dihydroxyvitamin D3: Biological activity of 1β,25-dihydroxyvitamin D3

The simple three-step preparation of [1β-³H]1α,25-dihydroxyvitamin D₃ and [1α-³H]1β,25-dihydroxyvitamin D₃ from 1α,25-dihydroxyvitamin D₃ is described. In the rat, 1β,25-dihydroxyvitamin D₃, when compared with its α-epimer, did not stimulate intestinal calcium transport or bone calcium mobilization at doses 1000-fold higher than the doses of the natural hormone, 1α,25-dihydroxyvitamin D₃.     

Metabolism of 1α,25-dihydroxyvitamin D2 by human CYP24A1

The metabolism of 1α,25-dihydroxyvitamin D₂ (1α,25(OH)₂D₂) by human CYP24A1 was examined using the recombinant enzyme expressed in Escherichia coli cells. HPLC analysis revealed that human CYP24A1 produces at least 10 metabolites, while rat CYP24A1 produces only three metabolites, indicating a remarkable species-based difference in the CYP24A1-dependent metabolism of 1α,25(OH)₂D₂ between humans and rats. LC-MS analysis and periodate treatment of the metabolites strongly suggest that human CYP24A1 converts 1α,25(OH)₂D₂ to 1α,24,25,26(OH)₄D₂, 1α,24,25,28(OH)₄D₂, and 24-oxo-25,26,27-trinor-1α(OH)D₂ via 1α,24,25(OH)₃D₂. These results indicate that human CYP24A1 catalyzes the C₂₄-C₂₅ bond cleavage of 1α,24,25(OH)₂D₂, which is quite effective in the inactivation of the active form of vitamin D₂. The combination of hydroxylation at multiple sites and C-C bond cleavage could form a large number of metabolites. Our findings appear to be useful to predict the metabolism of vitamin D₂ and its analogs in the human body.     

Extremely high circulating levels of 1α,25-dihydroxyvitamin D3 in the marmoset,a new world monkey

Compared to most mammals, the marmoset, a new world monkey, requires particularly large amounts of vitamin D to maintain normal growth. We compared serum concentrations of vitamin D metabolites in marmosets with rhesus monkeys and humans. The circulating levels of 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] in marmosets were 4 to 10 times higher than those in rhesus monkeys and humans. But none of the marmosets exhibited hypercalcemia. In two marmosets which had suffered bone fractures, the 1α,25-(OH)₂D₃ levels were particularly elevated. These results suggest that the marmoset has an end-organ resistance to 1α,25(OH)₂D₃.     

A new insight into the role of rat cytochrome P450 24A1 in metabolism of selective analogs of 1α,25-dihydroxyvitamin D3

We examined the metabolism of two synthetic analogs of 1α,25-dihydroxyvitamin D₃ (1), namely 1α,25-dihydroxy-16-ene-23-yne-vitamin D₃ (2) and 1α,25-dihydroxy-16-ene-23-yne-26,27-dimethyl-vitamin D₃ (4) using rat cytochrome P450 24A1 (CYP24A1) in a reconstituted system. We noted that 2 is metabolized into a single metabolite identified as C26-hydroxy-2 while 4 is metabolized into two metabolites, identified as C26-hydroxy-4 and C26a-hydroxy-4. The structural modification of adding methyl groups to the side chain of 1 as in 4 is also featured in another analog, 1α,25-dihydroxy-22,24-diene-24,26,27-trihomo-vitamin D₃ (6). In a previous study, 6 was shown to be metabolized exactly like 4, however, the enzyme responsible for its metabolism was found to be not CYP24A1. To gain a better insight into the structural determinants for substrate recognition of different analogs, we performed an in silico docking analysis using the crystal structure of rat CYP24A1 that had been solved for the substrate-free open form. Whereas analogs 2 and 4 docked similar to 1, 6 showed altered interactions for both the A-ring and side chain, despite prototypical recognition of the CD-ring. These findings hint that CYP24A1 metabolizes selectively different analogs of 1, based on their ability to generate discrete recognition cues required to close the enzyme and trigger the catalytic mechanism.     

Determination of 22-oxacalcitriol, a new analog of 1α,25-dihydroxyvitamin D3, in human serum by liquid chromatography–mass spectrometry

A sensitive and specific liquid chromatographic–mass spectrometric assay has been developed for the determination of 22-oxacalcitriol (OCT), which is a new analog of 1α,25-dihydroxyvitamin D₃. The analyte was isolated from serum by two solid-phase extraction steps on a C₁₈ cartridge and NH₂ cartridge. The recovery of OCT through two extraction steps was more than 90%. A related substance (ED-94), i.e. OCT with the side-chain shortened by one carbon, was used as an internal standard. Extracts were chromatographed on a C₁₈ reversed-phase column interfaced to the electrospray ionization source. The mass spectrometer was operated in the positive-ion mode of selected reaction monitoring. The chromatographic run-time for one injection was less than 6 min. The intra- and inter-assay coefficients of variation for the lowest concentration examined (30 pg ml⁻¹) were 9.83 and 10.67, respectively. And the analytical recovery of OCT added to serum was quantitative. Assay linearity was obtained in the range of 20–640 pg ml⁻¹.     

1α,25-Dihydroxyvitamin D3; its role for homeostasis of keratinocytes

Epidermal homeostasis is influenced by a number of hormones and regulative growth factors that are maintained by a tightly regulated balance between cell proliferation, cell differentiation, and cell death. Vitamin D is one of those regulatory factors. It has recently been demonstrated that 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃) takes part in the regulation of the cell cycle by multiple and complex functions. This review discusses 1α,25(OH)₂D₃ and its analogues in connection with the renewal of epidermal keratinocytes as well as the molecular mechanisms underlying terminal differentiation or rather programmed cell death. Furthermore, interest is focused on the possible clinical application of vitamin D₃ analogues.     

1α,25-Dihydroxyvitamin D3 induces differentiation of human myeloid leukemia cells

A human myeloid leukemia cell line [HL-60] could be induced to differentiate into mature myeloid cells by 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃], the active form of vitamin D₃. At 10^?10–10^?8 M, 1α,25(OH)₂D₃ suppressed cell growth in a dose-dependent manner and markedly induced phagocytosis and C3 rosette formation. The potency of 1α,25(OH)₂D₃ in inducing differentiation was nearly equivalent to that of known synthetic inducers such as dimethyl sulfoxide, actinomycin D or a phorbol ester (12-o-tetra-decanoyl-phorbol-13-acetate). These results clearly indicate that 1α,25(OH)₂D₃, besides its well known biological effect in enhancing intestinal calcium transport and bone mineral mobilization activities, is involved in the cell grwoth and differentiation of HL-60 cells.     

Does estradiol stimulate in vivo production of 1,25-dihydroxyvitamin D3 in the rat?

Female rats were injected with estradiol benzoate (3 mg/kg daily) or corn oil for 8 days before receiving 325 picomoles of 25-[26,27³H]-hydroxyvitamin D₃ intravenously. Eighteen hours later lipids were extracted from plasma, gut mucosa and kidneys and vitamin D₃ metabolites were separated using Sephadex LH-20 and chloroform: hexanes (65:35) column chromatography. Estradiol treatment increased 1,25-[26,27-³H]-dihydroxyvitamin D₃ content in all three isssues.     

Transformation of vitamin D3 to 1α,25-dihydroxyvitamin D3 via 25-hydroxyvitamin D3 using Amycolata sp. strains

To enzymatically synthesize active metabolites of vitamin D₃, we screened about 500 bacterial strains and 450 fungal strains, of which 12 strains were able to convert vitamin D₃ to 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] via 25-hyroxyvitamin D₃ [25(OH)D₃]. The conversion activity was only detected in strains belonging to the genus Amycolata among all the organisms tested. A preparative-scale conversion of vitamin D₃ to 25(OH)D₃ and 1,25(OH)₂D₃ in a 200-1 tank fermentor using A. autotrophica FERM BP-1573 was accomplished, yielding 8.3 mg 25(OH)D₃/l culture and 0.17 mg 1,25(OH)₂D₃/l culture. A related compound, vitamin D₂, could be also converted to 25-hydroxyvitamin D₂ and 1,25-dihydroxyvitamin D₂ using the same strain. The cytochrome P-450 of FERM BP-1573 was detected by reduced CO difference spectra in whole-cell suspensions. Vitamin D₃ in the culture induced cytochrome P-450 and the conversion activity simultaneously, suggesting that the hydroxylation at C-25 of vitamin D₃ and at C-1 of 25(OH)D₃ originates from cytochrome P-450.Correspondence to: J. Sasaki     

Studies on the analysis of 25-hydroxyvitamin D3 by isotope-dilution liquid chromatography–tandem mass spectrometry using enzyme-assisted derivatisation

The total serum concentration of 25-hydroxyvitamins D (25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₂) is currently used as an indicator of vitamins D status. Vitamins D insufficiency is claimed to be associated with multiple diseases, thus accurate and precise reference methods for the quantification of 25-hydroxyvitamins D are needed. Here we present a novel enzyme-assisted derivatisation method for the analysis of vitamins D metabolites in adult serum utilising 25-[26,26,26,27,27,27-²H₆]hydroxyvitamin D₃ as the internal standard. Extraction of 25-hydroxyvitamins D from serum is performed with acetonitrile, which is shown to be more efficient than ethanol. Cholesterol oxidase is used to oxidize the 3β-hydroxy group in the vitamins D metabolites followed by derivatisation of the newly formed 3-oxo group with Girard P reagent. 17β-Hydroxysteroid dehydrogenase type 10 is shown to oxidize selectively the 3α-hydroxy group in the 3α-hydroxy epimer of 25-hydroxyvitamin D₃. Quantification is achieved by isotope-dilution liquid chromatography–tandem mass spectrometry. Recovery experiments for 25-hydroxyvitamin D₃ performed on adult human serum give recovery of 102–106%. Furthermore in addition to 25-hydroxyvitamin D₃, 24,25-dihydroxyvitamin D₃ and other uncharacterised dihydroxy metabolites, were detected in adult human serum.     

Synthesis of 1α-[2-H]Hydroxyvitamin D3

1α-[2-³H]Hydroxyvitamin D₃ was synthesized chemically. The preparation was radiochemically pure and had a high enough specific activity (4.2 Ci/mmol) to permit experiments using 62.5 pmol/rat. This preparation had as much effect as synthetic 1α-hydroxyvitamin D₃ in increasing the serum Ca level of vitamin D-deficient rats.     

The role of the vitamin D receptor and ERp57 in photoprotection by 1α,25-dihydroxyvitamin D3

UV radiation (UVR) is essential for formation of vitamin D(3), which can be hydroxylated locally in the skin to 1α,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)]. Recent studies implicate 1,25-(OH)(2)D(3) in reduction of UVR-induced DNA damage, particularly thymine dimers. There is evidence that photoprotection occurs through the steroid nongenomic pathway for 1,25-(OH)(2)D(3) action. In the current study, we tested the involvement of the classical vitamin D receptor (VDR) and the endoplasmic reticulum stress protein 57 (ERp57), in the mechanisms of photoprotection. The protective effects of 1,25-(OH)(2)D(3) against thymine dimers were abolished in fibroblasts from patients with hereditary vitamin D-resistant rickets that expressed no VDR protein, indicating that the VDR is essential for photoprotection. Photoprotection remained in hereditary vitamin D-resistant rickets fibroblasts expressing a VDR with a defective DNA-binding domain or a mutation in helix H1 of the classical ligand-binding domain, both defects resulting in a failure to mediate genomic responses, implicating nongenomic responses for photoprotection. Ab099, a neutralizing antibody to ERp57, and ERp57 small interfering RNA completely blocked protection against thymine dimers in normal fibroblasts. Co-IP studies showed that the VDR and ERp57 interact in nonnuclear extracts of fibroblasts. 1,25-(OH)(2)D(3) up-regulated expression of the tumor suppressor p53 in normal fibroblasts. This up-regulation of p53, however, was observed in all mutant fibroblasts, including those with no VDR, and with Ab099; therefore, VDR and ERp57 are not essential for p53 regulation. The data implicate the VDR and ERp57 as critical components for actions of 1,25-(OH)(2)D(3) against DNA damage, but the VDR does not require normal DNA binding or classical ligand binding to mediate photoprotection.     

Role of 1α,25(OH)2 vitamin D3 on α-[1-C]MeAIB accumulation in immature rat testis

1,25D₃ is critical for the maintenance of normal reproduction since reduced fertility is observed in male rats on a vitamin D-deficient diet. Vitamin D-deficient male rats have incomplete spermatogenesis and degenerative testicular changes. In the present study we have examined the ionic involvement and intracellular messengers of the stimulatory effect of 1,25D₃ on amino acid accumulation in immature rat testis. 1,25D₃ stimulates amino acid accumulation from 10⁻¹² to 10⁻⁶ M by increasing the slope to reach a maximum value at 10⁻¹⁰ M, as compared to the control group. No effect was observed at a lower dose (10⁻¹³ M). Time-course showed an increase on amino acid accumulation after 15, 30, and 60 min of incubation with 1,25D₃ (10⁻¹⁰ M). 1,25D₃ stimulated amino acid accumulation in 11-day-old rat testis but not in testis that were 20 days old. Cycloheximide totally blocked the 1,25D₃ action on amino acid accumulation. Furthermore, a localized elevation of cAMP increased the stimulatory effect of 1,25D₃ and the blockage of PKA nullified the action of the hormone. In addition, 1,25D₃ action on amino acid accumulation was also mediated by ionic pathways, since verapamil and apamine diminished the hormone effect. The stimulatory effect of 1,25D₃ on amino acid accumulation is age-dependent and specific to this steroidal hormone since testosterone was not able to change amino acid accumulation in both ages studied. This study provides evidence for a dual effect for 1,25D₃, pointing to a genomic effect that can be triggered by PKA, as well as to a rapid response involving Ca²⁺/K⁺ channels on the plasma membrane.     

1α,25-Dihydroxyvitamin D3 affects hormone production and expression of steroidogenic enzymes in human adrenocortical NCI-H295R cells

The current study presents data indicating that 1α,25-dihydroxyvitamin D₃ affects the production of hormones and expression of crucial steroidogenic enzymes in the human adrenocortical cell line NCI-H295R. This cell line is widely used as a model for adrenal steroidogenesis. Treatment of the cells with 1α,25-dihydroxyvitamin D₃ suppressed the levels of corticosterone, aldosterone, DHEA, DHEA-sulfate and androstenedione in the culture medium. In order to study the mechanisms behind this suppression of hormone production, we investigated the effects of 1α,25-dihydroxyvitamin D₃ on important genes and enzymes controlling the biosynthesis of adrenal hormones. The mRNA levels were decreased for CYP21A2 while they were increased for CYP11A1 and CYP17A1. No significant changes were observed in mRNA for CYP11B1, CYP11B2 or 3β-hydroxysteroid dehydrogenase (3βHSD). In similarity with the effects on mRNA levels, also the endogenous enzyme activity of CYP21A2 decreased after treatment with 1α,25-dihydroxyvitamin D₃. Interestingly, the two CYP17A1-mediated activities were influenced reciprocally — the 17α-hydroxylase activity increased whereas the 17,20-lyase activity decreased. The current data indicate that the 1α,25-dihydroxyvitamin D₃-mediated decrease in corticosterone and androgen production is due to suppression of the 21-hydroxylase activity by CYP21A2 and the 17,20-lyase activity by CYP17A1, respectively. In conclusion, the current study reports novel findings on 1α,25-dihydroxyvitamin D₃-mediated effects on hormone production and regulation of genes and enzymes involved in steroidogenesis in the adrenocortical NCI-H295R cell line, a model for human adrenal cortex.     

Combinatorial effect of fish oil (Maxepa) and 1α,25-dihydroxyvitamin D3 in the chemoprevention of DMBA-induced mammary carcinogenesis in rats

The present study demonstrates the anti-tumor effects of combined supplementations of dietary fish oil (Maxepa) and 1α,25-dihydroxyvitamin D₃ (vitamin D₃) on 7,12-dimethylbenz(α)anthracene (DMBA)-induced rat mammary carcinogenesis. Female Sprague–Dawley rats at 50 days of age were treated with 7,12-dimethylbenz(α)anthracene (DMBA; 0.5 mg/100 g body weight) by a single tail vein injection in an oil emulsion. Both fish oil (rich in EPA and DHA) and vitamin D₃ were administered orally at a dose of 0.5 ml/day/rat and 0.3 μg/100 μL propylene glycol twice a week respectively and continued to 35 weeks after DMBA administration. Fish oil in combination with vitamin D₃ resulted in a significant reduction in incidence, multiplicity and volume of mammary tumors. These supplementation also inhibited DMBA-induced mammary 7-methylguanine DNA adducts formation, which was measured by HPLC-fluorescence assay (at four sequential time points; ANOVA, F = 42.56, P < 0.0001). Immunohistochemical analysis revealed that the effect of fish oil and vitamin D₃ occurred through suppression of cell proliferation (BrdU-LI: P < 0.0001). Fish oil and vitamin D₃ together also reduced the mRNA expression of iNOS (84%, P < 0.05). In view of their natural availability, non-toxicity and acceptability; combined supplementation of fish oil and vitamin D₃ might be effective for chemoprevention of mammary carcinogenesis.