Formation of 1,24-24-trihydroxyvitamin D3 from 1,25-dihydroxyvitamin D3

Incubation of [26,27-³H₂]-25-hydroxyvitamin D₃ with kidney homogenates from rats fed a high (3%) calcium vitamin D-supplemented diet results in the production of a more polar metabolite which cochromatographs with 1,24,25-trihydroxyvitamin D₃. On the other hand, incubation with kidney homogenates from vitamin D-deficient or calcium-deficient rats did not produce the polar metabolite. Mitochondria but not microsomes carry out the reaction and evidence has been produced to demonstrate that the 1,24,25-trihydroxyvitamin D₃ can be produced in vivo from either 1,25-dihydroxyvitamin D₃ as previously reported.     

24R,25-dihydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3 are both indispensable for calcium and phosphorus homeostasis

The essential role of vitamin D throughout the life of most mammals and birds as a mediator of calcium homeostasis is well established. In view of the complex endocrine system existent for the regulated metabolism of vitamin D₃ to both 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] and 24R,25-dihydroxyvitamin D₃ [24R,25-(OH)₂D₃] (both produced by the kidney), an intriguing problem is to elucidate whether only one or both of these dihydroxyvitamin D₃ metabolites is required for the generation of all the biological responses mediated by the parent vitamin D₃. In contrast to the accumulated knowledge concerning the short term actions of 1,25(OH)₂-D₃ on stimulating intestinal calcium absorption and bone calcium reabsorption, relatively little is known of the biological function of 24,25(OH)₂D₃. We report now the results of a nine month study in which chicks were raised on a vitamin D-deficient diet from hatching to sexual maturity and received as their sole source of “vitamin D” either 24,25(OH)₂D₃ or 1,25(OH)₂D₃ singly or in combination. Specifically we are describing the integrated operation of the vitamin D endocrine system as quantitated by the individual measurement in all birds of 22 variables related to “vitamin D status” and as evaluated by the statistical procedure of multivariate discriminant analysis. Twelve of these variables involved detailed analysis of the bone including quantitative histology and the other 10 variables reflect various manifestations of vitamin D action, e.g. serum Ca²⁺ and Pi levels, vitamin D-dependent calcium binding protein (CaBP) in the intestine and kidney, egg productivity etc. As evaluated by the multivariate analysis, it is clear that 24,25(OH)₂D₃ and 1,25(OH)₂D₃ are simultaneously required for normalization of calcium homeostasis.     

1,25,26-trihydroxyvitamin D3: isolation, identification, and biological activity

A polar metabolite of vitamin D₃ has been produced in vitro from either 1,25-dihydroxyvitamin D₃ incubated with kidney homogenate from vitamin D-supplemented chickens or from 25,26-dihydroxyvitamin D₃ incubated with vitamin D-deficient chicken kidney homogenate. This compound was isolated in pure form and identified as 1,25,26-trihydroxyvitamin D₃ by ultraviolet absorption spectrophotometry and mass spectrometry. Furthermore, its periodate cleavage product comigrates with synthetic 1α-hydroxy-25-keto-27-norvitamin D₃ on high-performance liquid chromatography. The 1,25,26-trihydroxyvitamin D₃ is 0.1-0.01 as active as 1,25-dihydroxyvitamin D₃ in the stimulation of intestinal calcium transport and bone calcium mobilization.     

Inhibitors of the 25-hydroxylation of vitamin D3 in the rat

Two new sidechain-modified analogs of vitamin D₃, 25-azavitamin D₃ and 25-fluorovitamin D₃, were prepared; both compounds were found to inhibit the in vivo 25-hydroxylation of vitamin D₃ in the rat. 25-Azavitamin D₃ was chemically synthesized from a degradation product of stigmasterol by a six-step process. The desired carbon skeleton was efficiently assembled by alkylation of a suitably protected C-20 bromomethylpregnane with the enolate of N,N-dimethylacetamide (70%). The completion of the synthesis utilized the known photochemistry of steroidal 5,7-dienes to prepare the vitamin D triene system. In contrast, 25-fluorovitamin D₃ was prepared by direct vitamin modification. 25-Hydroxyvitamin D₃ 3-acetate was fluorinated with diethylaminosulfur trifluoride to give 25-fluorovitamin D₃ 3-acetate (59%); saponification provided the desired analog. When vitamin D-deficient rats on a low calcium diet were dosed with [3-³H]vitamin D₃ (0.05 μg), 10% of the dose was found in serum as 25-hydroxyvitamin D₃ 4 hr after administration. If 25-azavitamin D₃ (50 or 200 μg) was given 2 hr before the radiolabeled vitamin D₃, however, serum 25-hydroxyvitamin D₃ concentration was markedly reduced. 25-Fluorovitamin D₃ caused similar reduction when administered at much lower doses.     

The synthesis of [26,27-C]dihydroxyvitamin D3, a tracer for positron emission tomography (PET)

1α,25-Dihydroxyvitamin D₃, an endogenous ligand with the highest affinity for the vitamin D receptor (VDR), was labeled with ¹¹C for use in biological experiments. The radionuclide was incorporated via the reaction of [¹¹C]methyllithium on a methyl ketone precursor in tetrahydrofuran at −10 °C. Deprotection of the labeled intermediate yielded 2.5–3 GBq [26,27-¹¹C]1α,25-dihydroxyvitamin D₃ [¹¹C-1,25(OH)₂ D₃] with specific radioactivity averaging 100 GBq/μmol at the end of synthesis and HPLC purification. The entire process took 48 min from the end of radionuclide production. In vitro binding experiments in rachitic chick purified VDR demonstrated the high affinity binding of this novel tracer. Thus; ¹¹C-1,25(OH)₂ D₃ is available for in vivo distribution studies and may be suitable for the positron emission tomography (PET) determination of VDR levels and occupancy in animals and humans.     

1α,25-Dihydroxyvitamin D3 stimulates colony formation of chick embryo chondrocytes in soft agar

The effect of vitamin D metabolites on the growth of chick embryo chondrocytes in soft agar was examined. 1,25-Dihydroxyvitamin D₃ [1,25(OH)₂D₃]at 10⁻⁸-10⁻⁷ M induced colony formation by chick embryo chondrocytes in soft agar in the presence of 10% fetal bovine serum. Furthermore, 1,25(OH)₂D₃ increased the number of colonies in the presence of a maximal dose of basic fibroblast growth factor, a potent mitogen for chondrocytes in soft agar. However, 24R,25 (OH)₂D₃ and other metabolites had little effect on the soft agar growth of chondrocytes in the presence or absence of basic fibroblast growth factor. These results suggest that 1,25(OH)₂D₃ is an active metabolite which may be involved in supporting cartilage growth.     

Production in vitro of 1 alpha,25-dihydroxyvitamin D3-26,23-lactone from 1 alpha,25-dihydroxyvitamin D2 by rat small intestinal mucosa homogenates

The metabolism of 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] in the rat has been studied under both in vivo and in vitro conditions. A time course study of the appearance of 1α,25-dihydroxyvitamin D₃-26,23-lactone in the plasma following intravenous or oral administration of 1α,25(OH)₂D₃ suggests that the small intestine may take part in production of the 1α,25(OH)₂D₃-26,23-lactone. In an in vitro study using a homogenate of rat small intestinal mucosa, 1α,25(OH)₂D₃ undergoes further metabolism to give more polar metabolite(s) which comigrate with authentic 1α,24,25-trihydroxyvitamin D₃ [1α,24,25(OH)₃D₃] on Sephadex LH-20 column chromatography. The metabolic profile obtained after high-pressure liquid chromatography reveals two major classes of metabolites, designated Peaks X and Y. Peak X is an unidentified metabolite of 1α,25(OH)₂D₃. Peak Y is chromatographically identical with 1α,25-dihydroxyvitamin D₃-26,23-lactone which has been recently isolated from the plasma of rats and dogs as a major metabolite produced in vivo from either 1α,25(OH)₂D₃ or 1α-hydroxyvitamin D₃ (N. Ohnuma, K. Bannai, H. Yamaguchi, Y. Hashimoto, and A. W. Norman, 1980, Arch. Biochem. Biophys.204, 387). The enzyme activity which produces metabolites X and Y in the rat intestinal homogenates is induced in vitamin D-replete rats by pretreatment of the animals with intravenous 1.25 μg/kg doses of 1α,25-dihydroxyvitamin D₃, 6 to 8 h previously.     

Direct modulation of 25-hydroxyvitamin D3 hydroxylation in kidney tubules by 1,25-dihydroxyvitamin D3

Kidney tubules obtained from chicks fed a high-calcium low-phosphorus diet retained 25-hydroxyvitamin D₃-1-hydroxylase activity after a 10 h incubation in serum-free minimum essential medium. Inclusion of 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) in the medium prompted a suppression of 25-hydroxyvitamin D₃-1-hydroxylase and the induction of 25-hydroxyvitamin D₃-24-hydroxylase activities. The enzyme switch-over response could be prompted by 1.6 × 10^?7 M 1,25-dihydroxyvitamin D₃ and occurred within 6 h following treatment. Medium calcium appeared to augment the metabolite's switch-over action.     

Biphasic action of prostaglandin E2 on conversion of 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 in chick renal tubules

The effect of PGE₂ on the conversion of 25-hydroxyvitamin D₃ (25 OH D₃) to 1,25-dihydroxyvitamin D₃ (1,25- (OH) ₂D₃) by isolated renal tubules from vitamin D deficient chicks was studied under a variety of experimental conditions. In the absence of added vitamin D metabolites, PGE₂ (2 × 10⁻⁶M) caused an immediate inhibition of formation of 1,25-(OH) ₂D₃, followed by a delayed stimulation, apparent after 15 h exposure to PGE₂. Pretreatment of the tubules with 1,25-(OH) ₂D₃ prevented the immediate inhibitory action of PGE₂, and allowed the stimulation to be apparent after 4 h exposure to PGE₂. The cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1-methyl xanthine (IBMX) significantly stimulated the formation of 1,25-(OH) ₂D₃. PGE₂ significantly inhibited 1,25-(OH) ₂D₃ formation in tubules which had been stimulated by IBMX. PGE₂ stimulated the adenylate cyclase activity in a crude particulate fraction from the chick kidney, and raised cyclic adenosine 3′, 5′-monophosphate (cyclic AMP) levels in the renal tubules.It is concluded that PGE₂ can either stimulate or inhibit 1,25-(OH) ₂D₃ formation in chick renal tubules. The stimulatory effect may be partly due to elevation of cyclic AMP. The mechanism of the inhibitory effect requires further investigation.     

1α,25-dihydroxy-24-oxo-16-ene vitamin D3, a metabolite of a synthetic vitamin D3 analog, 1α,25-dihydroxy-16-ene vitamin D3, is equipotent to its parent in modulating growth and differentiation of human leukemic cells

1α,25(OH)₂-16-ene-D₃, a synthetic analog of the steroid hormone, 1α,25(OH)₂D₃, has great potential to become a drug in the treatment of leukemia and other proliferative disorders, because of its minimal in vivo calcemic activity associated with a potent inhibitory effect on cell growth. However, at present, the mechanisms through which 1α,25(OH)₂-16-ene-D₃ expresses its biological activities are still not completely understood. Our previous in vitro study in a perfused rat kidney indicated for the first time that 1α,25(OH)₂-16-ene-D₃ and 1α,25(OH)₂D₃ are metabolized differently. 1α,25(OH)₂-24-oxo-16-ene-D₃, an intermediary metabolite of 1α,25(OH)₂-16-ene-D₃ formed through the C-24 oxidation pathway, accumulated significantly in the perfusate when compared to 1α,25(OH)₂-24-oxo-D₃, the corresponding intermediary metabolite of 1α,25(OH)₂D₃. In a subsequent in vivo study, we also reported that 1α,25(OH)₂-24-oxo-16-ene-D₃ exerted immunosuppressive activity equal to its parent, without causing significant hypercalcemia. In order to establish further the critical role of 1α,25(OH)₂-24-oxo-16-ene-D₃, in generating some of the key biological activities ascribed to its parent, we performed the present in vitro study using a human myeloid leukemic cell line (RWLeu-4) as a model. Comparative target tissue metabolism studies indicated that 1α,25(OH)₂-16-ene-D₃ and 1α,25(OH)₂D₃ are metabolized differently in RWLeu-4 cells, and the differences were similar to the ones we previously observed in the rat kidney. The significant finding was the accumulation of 1α,25(OH)₂-24-oxo-16-ene-D₃ in RWLeu-4 cells because of its resistance to further metabolism. Biological activity studies indicated that both 1α,25(OH)₂-16-ene-D₃ and its 24-oxo metabolite produced growth inhibition and promoted differentiation of RWLeu-4 cells to the same extent, and these activities were several fold higher than those exerted by 1α,25(OH)₂D₃. In addition, the genomic action of each vitamin D compound was assessed in a rat osteosarcoma cell line (ROS 17/2.8) by measuring its ability to transactivate a gene construct containing the vitamin D response element of the osteocalcin gene linked to the growth hormone reporter gene. In these studies, both 1α,25(OH)₂-16-ene-D₃ and its 24-oxo metabolite exerted similar but potent transactivation activity which was several fold greater than that exerted by 1α,25(OH)₂D₃ itself. In summary, our results indicate that the production and slow clearance of the bioactive intermediary metabolite, 1α,25(OH)₂-24-oxo-16-ene-D₃, in RWLeu-4 cells contributes significantly to the final expression of the enhanced biological activities ascribed to its parent analog, 1α,25(OH)₂-16-ene-D₃.     

Ternary solvent mixtures for improved resolution of hydroxylated metabolites of vitamin D2 and vitamin D3 during high-performance liquid chromatography

This paper reports the development of three new ternary solvent mixtures for the liquid-chromatographic separation of metabolites of vitamin D on microparticulate silica. All solvent systems offer reduced peak tailing and improved resolution of vitamin D compounds, particularly of 24(R),25-(OH)₂D₃, when compared to the commonly used hexane—isopropanol mixture. The new mixtures can be substituted for hexane—isopropanol systems presently used for preparative liquid-chromatographic steps prior to radioimmunoassay or competitive protein-binding assay of 24,25-(OH)₂D and 1,25-(OH)₂D in human plasma. Hexane—isopropanol—methanol (87:10:3) mixtures are recommended where the lipid content of samples is high, whereas hexane—ethanol—chloroform (80:10:10) promises to be a useful mixture for differentiating vitamin D₃ metabolites from their vitamin D₂ analogs. A combination of the two solvent systems permits the separate assay of both 24(R),25-(OH)₂D₃ and 24(R),25-(OH)₂D₂ as well as 1,25-(OH)₂D₃ and 1,25-(OH)₂D₂.     

Circulating vitamin D3 and 25-hydroxyvitamin D in humans: An important tool to define adequate nutritional vitamin D status

Circulating 25-hydroxyvitamin D [25(OH)D] is generally considered the means by which we define nutritional vitamin D status. There is much debate, however, with respect to what a healthy minimum level of circulation 25(OH)D should be. Recent data using various biomarkers such as intact parathyroid hormone (PTH), intestinal calcium absorption, and skeletal density measurements suggest this minimum level to be 80 nmol (32 ng/mL). Surprisingly, the relationship between circulating vitamin D₃ and its metabolic product—25(OH)D₃ has not been studied. We investigated this relationship in two separate populations: the first, individuals from Hawaii who received significant sun exposure; the second, subjects from a lactation study who received up to 6400 IU vitamin D₃/day for 6 months.Results (1) the relationship between circulating vitamin D₃ and 25(OH)D in both groups was not linear, but appeared saturable and controlled; (2) optimal nutritional vitamin D status appeared to occur when molar ratios of circulating vitamin D₃ and 25(OH)D exceeded 0.3; at this point, the V_max of the 25-hydroxylase appeared to be achieved. This was achieved when circulating 25(OH)D exceeded 100 nmol.We hypothesize that as humans live today, the 25-hydroxylase operates well below its V_max because of chronic substrate deficiency, namely vitamin D₃. When humans are sun (or dietary) replete, the vitamin D endocrine system will function in a fashion as do these other steroid synthetic pathways, not limited by substrate. Thus, the relationship between circulating vitamin D and 25(OH)D may represent what “normal” vitamin D status should be.     

Synthesis and biological activity of 22-iodo- and (E)-20(22)-dehydro analogues of 1α,25-dihydroxyvitamin D3

Construction of 25-hydroxy-steroidal side chain substituted with iodine at C-22 was elaborated on a model PTAD-protected steroidal 5,7-diene and applied to a synthesis of (22R)- and (22S)-22-iodo-1α,25-dihydroxyvitamin D₃. Configuration at C-22 in the iodinated vitamins, obtained by nucleophilic substitution of the corresponding 22S-tosylates with sodium iodide, was determined by comparison of their iodine-displacement processes and cyclizations leading to isomeric five-membered (22,25)-epoxy-1α-hydroxyvitamin D₃ compounds. Also, 20(22)-dehydrosteroids have been obtained and their structures established by ¹H NMR spectroscopy. When compared to the natural hormone, (E)-20(22)-dehydro-1α,25-dihydroxyvitamin D₃ was found 4 times less potent in binding to the porcine intestinal vitamin D receptor (VDR) and 2 times less effective in differentiation of HL-60 cells. 22-Iodinated vitamin D analogues showed somewhat lower in vitro activity, whereas (22,25)-epoxy analogues were inactive. Interestingly, it was established that (22S)-22-iodo-1α,25-dihydroxyvitamin D₃ was 3 times more potent than its (22R)-isomer in binding to VDR and four times more effective in HL-60 cell differentiation assay. The restricted mobility of the side chain of both 22-iodinated vitamin D compounds was analyzed by a systematic conformational search indicating different spatial regions occupied by their 25-oxygen atoms. Preliminary data on the in vivo calcemic activity of the synthesized vitamin D analogues indicate that (E)-20(22)-dehydro-1α,25-dihydroxyvitamin D₃ and 22-iodo-1α,25-dihydroxyvitamin D₃ isomers were ca. ten times less potent than the natural hormone 1α,25-(OH)₂D₃ both in intestinal calcium transport and bone calcium mobilization.     

Effect of dietary calcium and phosphorus on intestinal calcium absorption and vitamin D metabolism.

To understand better dietary regulation of intestinal calcium absorption, a quantitative assessment of the metabolites in plasma and duodenum of rats given daily doses of radioactive vitamin D₃ and diets differing in calcium and phosphorus content was made. All known vitamin D metabolites were ultimately identified by high-pressure liquid chromatography. In addition to the known metabolites (25-hydroxyvitamin D₃, 24,25-dihydroxyvitamin D₃, 1,25-dihydroxyvitamin D₃, 25,26-dihydroxyvitamin D₃, and 1,24,25-trihydroxyvitamin D₃), several new and unidentified metabolites were found. In addition to 1,25-dihydroxyvitamin D₃ and 1,24,25-trihydroxyvitamin D₃, the levels of some of the unknown metabolites could be correlated with intestinal calcium transport. However, whether or not any of these metabolites plays a role in the stimulation of intestinal calcium absorption by low dietary calcium or low dietary phosphorus remains unknown.     

Use of vitamin D4 analogs to investigate differences in hepatic and target cell metabolism of vitamins D2 and D3

In this study, we used molecules with either of the structural differences in the side chains of vitamin D₂ and vitamin D₃ to investigate which feature is responsible for the significant differences in their respective metabolism, pharmacokinetics and toxicity. We used two cell model systems—HepG2 and HPK1A-ras—to study hepatic and target cell metabolism, respectively. Studies with HepG2 revealed that the pattern of 24- and 26-hydroxylation of the side chain reported for 1α-hydroxyvitamin D₂ (1α-OH-D₂) but not for 1α-OH-D₃ is also observed in both 1α-OH-D₄ and Δ²²-1α-OH-D₃ metabolism. This suggests that the structural feature responsible for targeting the enzyme to the C24 or C26 site could be either the C24 methyl group or the 22–23 double bond. In HPK1A-ras cells, the pattern of metabolism observed for the 24-methylated derivative, 1α,25-(OH)₂D₄, was the same pattern of multiple hydroxylations at C24, C26 and C28 seen for vitamin D₂ compounds without evidence of side chain cleavage observed for vitamin D₃ derivatives, suggesting that the C24 methyl group plays a major role in this difference in target cell metabolism of D₂ and D₃ compounds. Novel vitamin D₄ compounds were tested and found to be active in a variety of in vitro biological assays. We conclude that vitamin D₄ analogs and their metabolites offer valuable insights into vitamin D analog design, metabolic enzymes and maybe useful clinically.     

Tandem immunoaffinity chromatography for plasma 1α,25-dihydroxyvitamin D3 utilizing two antibodies having different specificities: A novel and powerful pretreatment tool for 1α,25-dihydroxyvitamin D3 radioreceptor assays

We report here a novel and powerful pretreatment method for radioreceptor assays (RRAs) for human plasma 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) based on “Tandem” immunoaffinity chromatography (Tandem IAC). Two antibodies having different specificities were each immobilized on agarose gel with cyanogen bromide to produce immunosorbents which were stable and repeatedly usable. An ethyl ether extract of plasma was applied to the first affinity column, from which 1,25(OH)₂D₃ could be preferentially eluted and separated from 1α-deoxy type metabolites. The effluent was then submitted to the second column, and the 1,25(OH)₂D₃ retained was eluted after non- or weakly-adsorbed interfering substances were washed out. This procedure allowed efficient purification without careful handling or strict time-management in the entire operation and enabled avoiding preparative high-performance liquid chromatography (HPLC) from RRA even with a conventional chick intestinal vitamin D receptor. Mean (± SD) plasma 1,25(OH)₂D₃ values of 56 normal subjects and 10 patients with chronic renal failure, obtained with this Tandem IAC/RRA system, were 36.4 (8.7) and 11.2 (4.0) pg/ml, respectively. The Tandem IAC will also be useful for developing immunoassays or gas chromatography-mass spectrometry of 1,25(OH)₂D₃.     

Purification, characterization, and directed evolution study of a vitamin D3 hydroxylase from Pseudonocardia autotrophica

Vitamin D₃ (VD₃) is a fat-soluble prohormone that plays a crucial role in bone metabolism, immunity, and control of cell proliferation and cell differentiation in mammals. The actinomycete Pseudonocardia autotrophica is capable of bioconversion of VD₃ into its physiologically active forms, namely, 25(OH)VD₃ or 1α,25(OH)₂VD₃. In this study, we isolated and characterized Vdh (vitamin D₃ hydroxylase), which hydroxylates VD₃ from P. autotrophica NBRC 12743. The vdh gene encodes a protein containing 403 amino acids with a molecular weight of 44,368 Da. This hydroxylase was found to be homologous with the P450 belonging to CYP107 family. Vdh had the same ratio of the V_max values for VD₃ 25-hydroxylation and 25(OH)VD₃ 1α-hydroxylation, while other enzymes showed preferential regio-specific hydroxylation on VD₃. We characterized a collection of Vdh mutants obtained by random mutagenesis and obtained a Vdh-K1 mutant by the combination of four amino acid substitutions. Vdh-K1 showed one-order higher VD₃ 25-hydroxylase activity than the wild-type enzyme. Biotransformation of VD₃ into 25(OH)VD₃ was successfully accomplished with a Vdh-expressed recombinant strain of actinobacterium Rhodococcus erythropolis. Vdh may be a useful enzyme for the production of physiologically active forms of VD₃ by a single cytochrome P450.     

The stereochemical configuration of the natural 23,25,26-trihydroxyvitamin D3

Four possible diastereoisomers of 23,25,26-trihydroxyvitamin D3 were synthesized and compared with the natural metabolite. The 4 synthetic diastereoisomers could be separated into 4 peaks by high-performance liquid chromatography. The natural 23,25,26-trihydroxyvitamin D3 comigrated with 23(S),25(R),26-trihydroxyvitamin D3. This result unequivocally demonstrates that the stereochemistry of the natural 23,25,26-trihydroxyvitamin D3 has the 23(S) and 25(R) configuration.