Isolation,identification, and biological activity of 23,25,26-trihydroxyvitamin D3, an in vitro and in vivo metabolite of vitamin D3

Kidney homogenates from vitamin D₃-supplemented chicks incubated with 25-hydroxyvitamin D₃ [25(OH)D₃] produce significant quantities of a new, unknown vitamin D metabolite. This metabolite was isolated in pure form from such incubation mixtures by using Sephadex LH-20 column chromatography followed by high-pressure liquid chromatography. This metabolite has been identified as 23,25,26-trihydroxyvitamin D₃ [23,25,26(OH)₃D₃] by loss of radioactivity from 25-hydroxy[23,24-³H]vitamin D₃ and 25-hydroxy-[26,27-methyl-³H]vitamin D₃, ultraviolet absorption spectrophotometry, mass spectrometry, and periodate cleavage oxidation followed by mass spectrometry. This same metabolite was also isolated from the serum of rats given large doses of vitamin D₃, and structurally characterized as 23,25,26-trihydroxyvitamin D₃. As yet, the stereochemistry at the C-23 and C-25 positions of the natural product remains unknown. A comparison of responses to a single dose level (500 ng) of 23,25,26(OH)₃D₃ or 25(OH)D₃ over 96 h in vitamin D-deficient rats indicated that the new metabolite had no capability to mediate bone calcium mobilization and that it was only weakly active in stimulating intestinal calcium transport.     

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.     

Metabolism of 25-hydroxy-vitamin D3 by primary cultures of chick kidney cells

The primary culture of kidney cells from vitamin D deficient chicks is described. After four days in culture the cells reach confluency and retain their ability to metabolize 25-hydroxyvitamin D₃ to 1,25-dihydroxyvitamin D₃. Addition of one unit of bovine parathyroid hormone to the culture medium for 48 hours prior to assay had no effect on the cells' ability to produce 1,25-dihydroxy vitamin D₃, whereas after 24 hours in the presence of 5×10^?8$\text{M}$ 1,25-dihydroxyvitamin D₃ the cells produced not this metabolite, but 24,25-dihydroxyvitamin D₃. This cell culture system will allow the investigation of the regulation of renal 25-hydroxyvitamin D₃ metabolism under controlled $\text{in vitro}$ conditions.     

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.     

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.     

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.     

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.     

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.     

Role of Vitamin D3 Receptor in the Synergistic Differentiation of WEHI-3B Leukemia Cells by Vitamin D3 and Retinoic Acid

WEHI-3B D⁻ cells differentiate in response to 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) but not to all-trans-retinoic acid (RA) or other inducing agents. Combinations of RA with 1,25-(OH)₂D₃ interact to produce synergistic differentiation of WEHI-3B D⁻ cells. To determine factors involved in the synergistic interaction, expression of the 1,25-(OH)₂D₃ receptor (VDR) and retinoid receptors, RARα and RXRα, was measured. No VDR was detected in untreated WEHI-3B D⁻ cells; however, RA and 1,25-(OH)₂D₃ when used as single agents caused a slight induction of the VDR and in combination produced a marked increase in the VDR. In contrast, no changes in RARα and RXRα were initiated by these compounds. An RAR-selective agonist combined with 1,25-(OH)₂D₃ produced synergistic differentiation of WEHI-3B D⁻ cells, whereas an RXR-selective agonist did not. To gain information on the role of the VDR in the synergistic interaction, the VDR gene was transferred into WEHI-3B D⁺ cells, in which no VDR was detected and no synergism was produced. Expression of the VDR conferred differentiation responsiveness to 1,25-(OH)₂D₃ in WEHI-3B D⁺ cells. These findings suggest that (a) induction of VDR expression is a key component in the synergistic differentiation induced by 1,25-(OH)₂D₃ and RA and (b) RAR and not RXR must be activated for enhanced induction of the VDR and for the synergistic differentiation produced by RA and 1,25-(OH)₂D₃.     

The clearance of human fibrinogen fragments D1, D2, D3 and fibrin fragment D1 dimer in mice

The clearance of human fibrinogen fragments D₁, D₂, D₃ and fibrin fragment D₁ dimer were studied in the mouse model. Clearance of these fragments is a complex process involving clearance from blood into three other compartments. The overall clearance of fragment D₁ and its dimer were essentially identical. Fragments D₂ and D₃ cleared at a progressively slower rate. Competition studies were performed between ¹²⁵I-labeled fragment D₁ and large molar excesses of unlabelled human fragments D₁, D₂, D₃, D₁ dimer, fragment E, fibrinogen, macroalbumin, mannan and asialooroscomucoid. Of these ligands only the fragment D variants competed for the clearance of ¹²⁵I-labeled fragment D₁. Cross-competition was observed when ¹²⁵I-labeled fragment D₁ dimer was cleared in the presence of large molar excesses of fragment D₁. Autopsies demonstrated that injected fragments D₁, D₂, D₃ and D₁ dimer cleared primarily in liver and kidneys. In some clearance studies, livers were perfused with tissue culture fluid, subjected to light microscopic autoradiography, and silver grain counts performed to localize cleared fragment D₁. These experiments indicated that 80% of the liver uptake was in hepatocytes. However, when silver grain counts were normalized for the number of parenchymal and nonparenchymal cells, the distribution of silver grains was essentially identical (1.8 and 1.6 grains per cell, respectively). It is concluded that fragments D₁, D₂, D₃ and D₁ dimer are recognized by a similar clearance pathway. Since neither fibrinogen nor fragment E competed for the clearance of fragment D₁, it is suggested that determinants present in the fragment D domain become exposed after plasmin attack on fibrinogen and are responsible for clearance.     

Effects of BMP-2 and vitamin D3 on the osteogenic differentiation of adipose stem cells

We studied the effect of bone morphogenetic protein-2 (BMP-2) and vitamin D₃ on the osteogenic differentiation of adipose stem cells (ASCs). ASCs were treated with 10, 50, and 100 ng/ml of BMP-2, and 10⁻⁸, 10⁻⁷, 10⁻⁶ M vitamin D₃. Then, to investigate the effects of combined treatment, ASCs were treated with BMP-2 and vitamin D₃ dose-dependently and time-dependently. The osteogenic differentiation was assessed by alkaline phosphatase (ALP) activities/staining and the mineralization was evaluated by Alizarin red S staining. ALP activity and mineralization dose-dependently increased in early stages (ALP on 7th day and mineralization on the 14th day) while all three doses of BMP-2 or vitamin D₃ showed comparable effects in late stages (ALP on the 14th day and mineralization on the 21st day) in ASCs. BMP-2 and vitamin D₃ had synergistic effect on the osteogenic differentiation of ASCs. While all three doses of BMP-2 acted similarly in reinforcing the effect of vitamin D₃, vitamin D₃ dose-dependently augmented the osteogenic effect of BMP-2. When BMP-2 was constantly treated, vitamin D₃ effect did not differ depending on the period of vitamin D₃ treatment. However, when vitamin D₃ was constantly treated, the BMP was more effective when treated for the last 7 days than when treated for the first 7 days. In conclusion, BMP-2 and vitamin D₃ promote osteogenic differentiation of ASCs, and can work synergistically. These results can be used to induce effective and economical osteogenic induction of ASCs for bone tissue engineering.     

Determination of 25-hydroxyvitamin D3 in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A method for the determination of 25-hydroxyvitamin D₃, the major metabolite of vitamin D₃ in human plasma, using a non-radioactive internal standard and reversed-phase high-performance liquid chromatography with UV detection (265 nm) has been developed. The method was applied to the determination of the metabolite in plasma from healthy subjects (n=25) and from patients with chronic renal failure (n=12). 25-Hydroxyvitamin D₃ 3-sulfate, a major conjugated metabolite of 25-hyroxyvitamin D₃, was also determined and the correlation between the concentrations of these metabolites was examined. The study showed that almost equal amounts of both compounds were detected in the plasma of healthy subjects, however, in two subjects, the amount of sulfate in the free form was found to be about twice as high as normally detected. In contrast, the free form was predominant in the plasma of patients with chronic renal failure and the sulfate was not detected in four patients.     

The biliary excretion of {3H}-25-hydroxyvitamin D3 following chronic ethanol administration in the rat

The biliary excretion of {³H}-25-hydroxyvitamin D₃ was studied in rats fed a diet containing 36% of total calories as ethanol and in pair-fed controls. {³H}-25-hydroxyvitamin D₃ was injected i.v. and the biliary appearance of {³H}-compounds studied over a period of 80 minutes. Choleresis was not affected by chronic ethanol feeding averaging 98.7±10.3 μl min^?1 kg^?1 in Controls and 101.8±8.7 μl min^?1 kg^?1 in ethanol-fed animals. The total biliary excretion of {³H} was found to be 29% higher in ethanol-treated than control rats and represented 6.3±0.4% and 4.9±0.4% (p<0.05) of the injected dose at the end of the experiment. The biliary excretion rate as well as the biliary concentration of {³H}-25-hydroxyvitamin D³ congeners were significantly increased by the ethanol treatment from the 25th to the 80th minute after the i.v. injection of {³H}-25-hydroxyvitamin D₃. The plasma and liver {³H} concentrations were not significantly affected by the ethanol treatment. These results suggest that, in the rat, chronic ethanol ingestion promotes the biliary loss of {³H}-25-hydroxyvitamin D₃. The higher concentration of {³H}-compounds into the bile of ethanol treated rats also suggests the presence of excretory pathway(s) which could be, at least in part, independent of bile flow. This increased loss of {³H}-25-hydroxyvitamin D³ into the bile may be a contributing factor in the impaired vitamin D status in alcoholics.     

In vivo effects of chronic contamination with depleted uranium on vitamin D3 metabolism in rat

The extensive use of depleted uranium (DU) in today's society results in the increase of the number of human population exposed to this radionuclide. The aim of this work was to investigate in vivo the effects of a chronic exposure to DU on vitamin D₃ metabolism, a hormone essential in mineral and bone homeostasis. The experiments were carried out in rats after a chronic contamination for 9 months by DU through drinking water at 40 mg/L (1 mg/rat/day). This dose corresponds to the double of highest concentration found naturally in Finland. In DU-exposed rats, the active vitamin D (1,25(OH)₂D₃) plasma level was significantly decreased. In kidney, a decreased gene expression was observed for cyp24a1, as well as for vdr and rxrα, the principal regulators of CYP24A1. Similarly, mRNA levels of vitamin D target genes ecac1, cabp-d28k and ncx-1, involved in renal calcium transport were decreased in kidney. In the brain lower levels of messengers were observed for cyp27a1 as well as for lxrβ, involved in its regulation. In conclusion, this study showed for the first time that DU affects both the vitamin D active form (1,25(OH)₂D₃) level and the vitamin D receptor expression, and consequently could modulate the expression of cyp24a1 and vitamin D target genes involved in calcium homeostasis.     

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.     

1 Alpha-25,26-trihydroxyvitamin D3: an in vivo and in vitro metabolite of vitamin D3

A new metabolite of vitamin D3 has been isolated from the plasma of vitamin D3 treated cows and has been generated from 25(S),26-dihydroxyvitamin D3 with homogenates of vitamin D deficient chick kidney. This metabolite has been identified as 1,25,26-trihydroxyvitamin D3 by comigration with synthetic 1,25(S),26-trihydroxyvitamin D3 in four chromatographic systems, ultraviolet spectroscopy, mass spectrometry, and high-pressure liquid chromatography and mass spectrometry of derivatives. 1,25(S),26-Trihydroxyvitamin D3 is one-tenth as effective as 1,25-dihydroxyvitamin D3 in binding to the chick intestinal cytosol 1,25-dihydroxyvitamin D receptor. Either 25(S),26-dihydroxyvitamin D3 or 1,25-dihydroxyvitamin D3 can serve as precursor for in vitro production of 1,25,26-trihydroxyvitamin D3 by chick kidney tissue.     

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.     

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.