Sarcoid granulomas metabolize 25-hydroxyvitamin D3invitro

Sarcoid granulomas metabolized 25-hydroxyvitamin D₃ to two unidentified metabolites during $\text{in}\text{vitro}$ incubation. A two-step high pressure liquid chromatography system revealed two unique elution positions of these sarcoid-derived metabolites that exactly comigrated with the elution positions of 5(Z)-19-nor-10-oxo-25(OH)D₃ and 5(E)-19-nor-10-oxo-25(OH)D₃, respectively. These unique metabolites did not bind specifically to a protein receptor for 1,25(OH)₂D₃.     

Isolation, identification, and chemical synthesis of 8 alpha,25-dihydroxy-9,10-seco-4,6,10(19)-cholestatrien-3-one. A new metabolite of 25-hydroxyvitamin D3 produced by mouse myeloid leukemia cells (M1)

It is known that phagocytic cells such as monocyte-macrophages and myeloid leukemia cells metabolize 25-hydroxyvitamin D3 to 10-oxo-19-nor-25-hydroxyvitamin D3. Now we have found that phagocytic cells metabolize 25-hydroxyvitamin D3 not only to 10-oxo-19-nor-25-hydroxyvitamin D3 but also to a new metabolite eluted just after 24R,25-dihydroxyvitamin D3 on straight phase high pressure liquid chromatography with a 2-propanol-hexane solvent system. The new metabolite, produced by murine myeloid leukemia cells (M1), was isolated in pure form and identified as 8,25-dihydroxy-9,10-seco-4,6,10(19)-cholestatrien-3-one on the basis of mass, ultraviolet, infrared, and proton magnetic resonance spectra. The 8 alpha-hydroxy epimer of the putative metabolite was chemically synthesized in two steps starting from 25-hydroxyvitamin D3. The spectral data and chromatographic behavior of chemically synthesized 8 alpha,25-dihydroxy-9,10-seco-4,6,10(19)-cholestatrien-3-one coincided exactly with those of the isolated metabolite, indicating that the stereochemistry of the hydroxyl group at the 8-position is alpha. On the basis of the structural characteristics of the two metabolites produced from 25-hydroxyvitamin D3 (the present metabolite and 10-oxo-19-nor-25-hydroxyvitamin D3), it is suggested that dioxygenases are involved in the production of these metabolites from 25-hydroxyvitamin D3 in phagocytic cells.     

Metabolism of 25-hydroxycholecalciferol in human promyelocytic leukemia cells (HL-60). Isolation and identification of (5Z)- and (5E)-19-nor-10-oxo-25-hydroxycholecalciferol

Human promyelocytic leukemia cells incubated with 25-hydroxy[26,27-methyl-3H] cholecalciferol (1 microCi) or non-radioactive 25-hydroxycholecalciferol (550 micrograms) produced significant quantities of two vitamin D3 metabolites. The two metabolites were isolated and purified by methanol chloroform extraction and a series of chromatographic procedures. The metabolite purification and elution positions on these columns were followed by radioactivity and their ultraviolet absorption at 310 nm. The two metabolites have been unequivocally identified as (5Z)- and (5E)-19-nor-10-oxo-25-hydroxycholecalciferol by ultraviolet absorption spectrophotometry, mass spectrometry, Fourier-transform infrared spectrophotometry and co-chromatography with synthetic compounds on a high-performance liquid chromatograph. (5E)- but not (5Z)-19-nor-10-oxo-25-hydroxycholecalciferol was able to induce HL-60 cell phenotypic and functional differentiation. However, these two metabolites of 25-hydroxycholecalciferol did not bind specifically to the chick intestinal 3.7 S. receptor protein for 1 alpha,25-dihydroxycholecalciferol. The precise biological role of these metabolites is as yet unclear.     

Phagocytic cells metabolize 25-hydroxyvitamin D3 to 10-oxo-19-nor-25-hydroxyvitamin D3 and a new metabolite, 8 alpha,25-dihydroxy-9,10-seco-4,6,10(19)-cholestatrien-3-one

The metabolism of 25-hydroxyvitamin D3 [25(OH)D3] was examined in several phagocytic cells including alveolar macrophages and myeloid leukemia cells (M1, HL-60 and U937). Phagocytic cells converted 25(OH)D3 to 10-oxo-19-nor-25-hydroxyvitamin D3 and a new metabolite. The former metabolite was dominant in shorter incubation periods (1 h), whereas the latter dominated over longer incubation periods (24 h). The new metabolite was produced from 25(OH)D3 directly but not through 10-oxo-19-nor-25-hydroxyvitamin D3. The new metabolite was unequivocally identified as 8 alpha,25-dihydroxy-9-10-seco-4,6,10(19)-cholestatrien-3-one. These results suggest that phagocytic cells somehow promote oxidation of the triene part of vitamin D compounds.     

On the specificity of vitamin D compounds binding to chick pig intestinal 1,25-dihydroxyvitamin D3 receptor

The binding activity of four vitamin D metabolites and/or analogs for the intestinal 1,25-dihydroxyvitamin D3 receptor was evaluated after incubation at 25 degrees C for 1 h or at 0-4 degrees C for 18 h. The incubation conditions, which had no effect on the binding of 1,25-dihydroxyvitamin D3, had a dramatic effect on the binding of 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3 and a small but reproducible effect on 24,25-dihydroxyvitamin D3 binding to receptor. Affinities 10- to 20-fold higher were obtained for 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3, and affinities 3-fold higher were obtained for 24,25-dihydroxyvitamin D3 at the 0-4 degrees C/18-h incubation. A comparison of intestinal receptor from chick and pig with nine vitamin D compounds showed no major differences between the two species. The relative affinity of the vitamin D analogs to compete with tritiated 1,25-dihydroxyvitamin D3 for the receptor in pig nuclear extract, expressed as ratios of the molar concentration required for 50% binding of the tritiated 1,25-dihydroxyvitamin D3 compared to nonradioactive 1,25-dihydroxyvitamin D3, are as follows: 1,25-dihydroxyvitamin D3 (1) = 1,25-dihydroxyvitamin D2 = 24-homo-1,25-dihydroxyvitamin D3 greater than 1,24,25-trihydroxyvitamin D3 (4) greater than 25-hydroxyvitamin D3 (21) = 10-oxo-19-nor-25-hydroxyvitamin D3 = 1 alpha-hydroxyvitamin D3 (37) greater than 24,25-dihydroxyvitamin D2 (257) much much greater than vitamin D3 (greater than 10(6)).     

Metabolism of the vitamin D3 derivative (24R)-hydroxycalcidiol by human promyelocytic leukemia cells (HL-60). Isolation and identification of (5Z) and (5E)-(24R)-19-nor-10-oxo-24-hydroxycalcidiol

Human promyelocytic leukemia cells (HL-60 cells) incubated with (24R)-hydroxy[26,27-methyl-3H]calcidiol (0.2 microCi) or non-radioactive (24R)-hydroxycalcidiol (370 micrograms) produced significant quantities of two new vitamin D3 (calciol) metabolites. The metabolites were isolated from HL-60 cell culture media by methanol/chloroform extraction and a series of chromatographic procedures. The two new metabolites were identified as (5Z)- and (5E)-(24R)-19-nor-10-oxo-24-hydroxycalcidiol by HPLC analysis, ultraviolet absorption spectrophotometry, mass spectrometry and Fourier-transform infrared spectrophotometry. According to the isolation and purification procedures, the total amounts of 3.04 micrograms (5Z)-(24R)-19-nor-10-oxo-24-hydroxycalcidiol (lambda max = 310 nm, epsilon = 17070 M-1 cm-1) and 8.89 micrograms (5E)-(24R)-19-nor-10-oxo-24-hydroxycalcidiol (lambda max = 312 nm, e = 24,500 M-1 cm-1) were calculated, assuming an Mr of 418. The activity of 19-nor-10-oxo-(24R)-hydroxycalcidiol to promote HL-60 cell differentiation was higher than the activity of the precursor (24R)-hydroxycalcidiol suggesting a possible biological action of this metabolite in HL-60 cells.     

Production of 10-oxo-19-nor-25-hydroxyvitamin D3 by solubilized kidney mitochondria from chick and rat

Cholate-solubilized chick kidney mitochondria that 1-hydroxylated 25-hydroxyvitamin-D3 (25-OH-D3) upon reconstitution also produced 10-oxo-19-nor-25-OH-D3, which co-eluted with 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3) on normal phase high performance liquid chromatography (HPLC) with hexane:propanol-2 (9:1), the traditional chromatographic system for isolating 1,25-(OH)2-D3. The 10-oxo derivative was separated from 1,25-(OH)2-D3 by normal phase HPLC with dichloromethane:propanol-2 (19:1) or by reverse phase HPLC with methanol:water (4:1). Unlike 1,25-(OH)2-D3 production, formation of 10-oxo-19-nor-25-OH-D3 did not require a source of reducing equivalents and was blocked by the antioxidants, diphenyl-rho-phenylenediamine, and butylated hydroxytoluene, implicating a free radical or peroxidative synthetic mechanism. Rat kidney mitochondria solubilized with cholate or with cholate and Emulgen 911 produced 10-oxo-19-nor-25-OH-D3 but no detectable 1 alpha,25-(OH)2-D3. These results stress the importance of careful identification of vitamin D metabolites produced in vitro and suggest the use of alternate chromatographic conditions for isolating 1,25-(OH)2-D3 or inclusion of antioxidants in the assay of solubilized 1 alpha-hydroxylase to eliminate contamination of 1,25-dihydroxyvitamin D3 with 10-oxo-19-nor-25-OH-D3.     

Characterization of 19-nor-10-oxo-25-hydroxyvitamin D3 production by solubilized chick kidney mitochondria and bovine serum albumin

The vitamin D3 metabolite obtained from the incubation of 3-[(cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO)-solubilized chick kidney mitochondria with 25-hydroxyvitamin D3 (25-OH-D3) was identified to be 5(E)-19-nor-10-oxo-25-hydroxyvitamin D3 (5(E)-19-nor). The production of 19-nor was dependent on time and on protein concentration, but was not dependent on the pH of the incubation. 19-Nor was not formed in the absence of protein or when protein had been heat-treated following detergent solubilization. 19-Nor was not further metabolized to any other product upon incubation with the CHAPSO-solubilized proteins. No 19-nor-10-oxo derivative of 1,25(OH)2D3 was formed when 1,25(OH)2D3 was used as substrate in the incubation. Kinetic analysis showed a substrate saturation with an apparent Vmax of about 4.1 pmol/min.mg and S0.5 of approximately 1.3 x 10(-6) M. The production of 19-nor was not restricted to the CHAPSO-soluble protein fraction of kidney mitochondria but was also found in both the CHAPSO-soluble and -insoluble fractions of chick liver mitochondria and CHAPSO-treated bovine serum albumin (BSA). 19-Nor production by detergent-treated BSA also showed saturation kinetics with a similar S0.5 and an apparent Vmax which was about 5-fold higher than that obtained with CHAPSO-solubilized mitochondria. The evidence suggests that the formation of 19-nor is not mediated by a traditional enzyme, but does require protein. A mechanism for the conversion of 25-OH-E3 to 19-nor is proposed, in which the naturally-occurring 5(Z)-25-OH-D3 substrate binds to protein, isomerizes to 5(E)-25-OH-D3 and is oxidized by hydrogen peroxide to 5(E)-19-nor via a dioxetane intermediate.     

Regulation of 25-hydroxyvitamin D3 metabolism in a human promyelocytic leukemia cell line (HL-60): 1,25-dihydroxyvitamin D3 stimulates the synthesis of 24,25-dihydroxyvitamin D3

The human promyelocytic leukemia cell line HL-60 undergoes macrophage-like differentiation after exposure to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the biologically active metabolite of vitamin D3. In the current study, we demonstrate that 1,25(OH)2D3 also regulates 25-hydroxyvitamin D3 [25(OH)D3] metabolism in HL-60 cells. The presence of 1,25(OH)2D3 in the culture medium of HL-60 cells stimulated the conversion of 7-10% of the substrate [25(OH)D3] to a more polar metabolite, which was identified as 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] from the elution positions on sequential HPLC systems and the sensitivity to periodate treatment. The HL-60 subclone HL-60 blast, which is unresponsive to 1,25(OH)2D3 in terms of differentiation, also responded to 1,25(OH)2D3 treatment with the production of 24,25(OH)2D3. Maximal stimulation of 24,25(OH)2D3-synthesis (approximately 7 pmol/5 X 10(6) cells) in HL-60 cells was noted with a 12-h exposure to 10(-9) M 1,25(OH)2D3. The ability of vitamin D3 metabolites other than 1,25(OH)2D3 to induce the synthesis of 24,25(OH)2D3 in HL-60 cells was, with the exception of 1 alpha-hydroxyvitamin D3, in correlation with their reported affinities for the specific 1,25(OH)2D3 receptor which is present in HL-60 cells. Treatment of HL-60 cells with phorbol diesters abolished the 1,25(OH)2D3 responsiveness, while treatment with dimethylsulfoxide and interferon gamma did not markedly alter the 25(OH)D3 metabolism of HL-60 cells. Small amounts (approximately 1% of substrate) of two 25(OH)D3 metabolites, which comigrated with 5(E)- and 5(Z)-19-nor-10-keto-25-hydroxyvitamin D3 on two HPLC solvent systems, were synthesized by HL-60 cells, independently from 1,25(OH)2D3 treatment or stage of cell differentiation. Our results indicate that 1,25(OH)2D3 influences 25(OH)D3 metabolism of HL-60 cells independently from its effects on cell differentiation.     

The biological assessment of vitamin D3 metabolites produced by rumen bacteria

Biological assays were performed to evaluate 10-oxo-19-nor-vitamin D3 (10-oxo-D3) and 5(E) 25-hydroxy-10-oxo-19-nor-vitamin D3 (25-OH-10-oxo-D3) two bacterial products of vitamin D3 (D3) and 25-hydroxyvitamin D3 (25-OHD3) metabolism, respectively. The 5(Z) and 5(E) isomers of 10-oxo-D3 were, respectively, 40- and 80-fold less active than D3 in stimulating Ca+2 absorption from the gut. 25-Hydroxy-10-oxo-D3 did not stimulate Ca+2 absorption. Only 5(Z) 10-oxo-D3 induced mobilization of bone Ca+2. In addition, both 10-oxo-D3 and 25-OH-10-oxo-D3 showed poor affinities for either the plasma D3-binding protein or the thymus 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor. 10-Keto-D3 exhibited a plasma half-life of only 6 min. This was a much shorter half-life than that exhibited by other vitamin D metabolites and was expected because of the poor affinity 10-oxo-D3 has for the plasma vitamin D binding protein. Bacterial metabolism of D3 deactivates the vitamin, which allows ruminants to tolerate relatively large oral doses of D3.     

In vitro metabolism of 19-nor-1alpha, 25-(OH)2D2 in cultured cell lines: inducible synthesis of lipid- and water-soluble metabolites

The active vitamin D analog, 19-nor-1alpha,25-dihydroxyvitamin D2 (19-nor-1alpha,25-(OH)2D2), has a similar structure to the natural vitamin D hormone, 1a,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3), but lacks the C10-19 methylene group and possesses an ergosterol/ vitamin D2 rather than a cholesterol/vitamin D3 side chain. We have used this analog to investigate whether any of these structural features has any effect upon the type and rate of in vitro metabolism observed. Using a vitamin D-target cell, the human keratinocyte, HPK1A-ras, we observed formation of a number of metabolites, three of which were purified by extensive HPLC and conclusively identified by a combination of GC-MS and chemical derivatization as 19-nor-1alpha,24,25-(OH) 3D2, 19-nor-1alpha,24,25,26-(OH) 4D2, and 19-nor-1alpha,24,25,28-(OH)4,D2. The first metabolite is probably a product of the vitamin D-inducible cytochrome P450, P450cc24 (CYP24), while the latter two metabolites are likely to be further metabolic products of 19-nor-1alpha,24,25-(OH)3D2. These hydroxylated metabolites resemble those identified by other workers as products of the metabolism of 1alpha,25-(OH)2D2 in the perfused rat kidney. It therefore appears from the similar metabolic fate of 19-nor-1alpha,25-(OH)2D2 and 1alpha,25-(OH)2D2 that the lack of the C10-19 methylene group has little effect upon the nature of the lipid-soluble metabolic products and the rate of formation of these products seems to be comparable to that of products of 1alpha,25-(OH)2D3 in vitamin D-target cells. We also found extensive metabolism of 19-nor-1alpha,25(OH)2D2 to water-soluble metabolites in HPK1A-ras, metabolites which remain unidentified at this time. When we incubated 19-nor-1alpha,25-(OH)2D2 with the liver cell line HepG2, we obtained only 19-nor-1alpha,24,25-(OH)3D2. We conclude that 19-nor-1alpha,25-(OH)2D2 is efficiently metabolized by both vitamin D-target cells and liver cells.     

Metabolism of 25-hydroxyvitamin D3 by rat kidney cells in culture: Isolation and identification of cis- and trans-19-nor-10-oxo-25-hydroxyvitamin D3

A primary confluent culture of epithelial cells from rat kidney has been developed. These cells possess a 3.2–3.4 S high-affinity, low-capacity binding protein for 1,25-dihydroxyvitamin D₃. They metabolize 25-hydroxyvitamin D₃ to at least five metabolites. Two have been identified as 1,25-dihydroxyvitamin D₃ and 24,25-dihydroxyvitamin D₃. Two others have been identified by means of physical data and cochromatography as trans 19-nor-10-oxo-25-hydroxyvitamin D₃ and the other as its cis isomer. These two “metabolites” have not been observed in vivo, but one of them (cis) comigrates with 1,25-dihydroxyvitamin D₃ on straight-phase high-performance liquid chromatography. Thus, mere cochromatography on high-performance liquid chromatography is not sufficient to identify critical vitamin D metabolites.     

Isolation and identification of 25-hydroxyvitamin D3-26,23-peroxylactone. A novel in vivo metabolite of vitamin D3

A new vitamin D3 metabolite was isolated in pure form (18.2 micrograms) from the serum of rats given large doses (two doses of 26 mumol/rat) of vitamin D3. The new metabolite has been unequivocally identified as 3 beta, 25-dihydroxy-9,10-seco-5,7,10(19)-cholestatrieno-26,23-peroxylactone by ultraviolet absorption spectrophotometry, Fourier transform infrared spectrophotometry, mass spectrometry, field desorption mass spectrometry, and specific chemical reaction with triphenyl phosphine. The stereochemical configuration at the C-23 and c-25 positions of the 25-hydroxyvitamin D3-26-23-peroxylactone was definitely determined to be the 23(S)25(R),25-hydroxyvitamin D3-26,23-peroxylactone is suggested for this metabolite. The isolation involved chloroform-methanol extraction and four column chromatographic procedures. The metabolite purification and elution position on these columns were followed by UV measurement at 264 nm. This metabolite was ultimately resolved from the previously known 25-hydroxyvitamin D3-26,23-lactone by high pressure liquid chromatography using a Zorbax Sil column. The 25-hydroxyvitamin D3-26,23-peroxylactone was converted upon storage at room temperature or -20 degrees C into the 25-hydroxyvitamin D3-26,23-lactone. Since under the conditions of this isolation only the 26,23-peroxylactone and no 26,23-lactone of 25-hydroxyvitamin D3 was present in the rat serum, this suggests that the 25-hydroxyvitamin D3-26,23-peroxylactone is the naturally occurring metabolite.     

Metabolic studies using recombinant escherichia coli cells producing rat mitochondrial CYP24 CYP24 can convert 1alpha,25-dihydroxyvitamin D3 to calcitroic acid.

Previously we expressed rat 25-hydroxyvitamin D3 24-hydroxylase (CYP24) cDNA in Escherichia coli JM109 and showed that CYP24 catalyses three-step monooxygenation towards 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3 [Akiyoshi-Shibata, M., Sakaki, T., Ohyama, Y., Noshiro, M., Okuda, K. & Yabusaki, Y. (1994) Eur. J. Biochem. 224, 335-343]. In this study, we demonstrate further oxidation by CYP24 including four- and six-step monooxygenation towards 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3, respectively. When the substrate 25-hydroxyvitamin D3 was added to a culture of recombinant E. coli, four metabolites, 24, 25-dihydroxyvitamin D3, 24-oxo-25-hydroxyvitamin D3, 24-oxo-23, 25-dihydroxyvitamin D3 and 24,25,26,27-tetranor-23-hydroxyvitamin D3 were observed. These results indicate that CYP24 catalyses at least four-step monooxygenation toward 25-hydroxyvitamin D3. Furthermore, in-vivo and in-vitro metabolic studies on 1alpha,25-dihydroxyvitamin D3 clearly indicated that CYP24 catalyses six-step monooxygenation to convert 1alpha,25-dihydroxyvitamin D3 into calcitroic acid which is known as a final metabolite of 1alpha,25-dihydroxyvitamin D3 for excretion in bile. These results strongly suggest that CYP24 is largely responsible for the metabolism of both 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3.     

Relationship of 25-hydroxyvitamin D3 side chain structure to biological activity.

27-nor-25-Hydroxyvitamin D3, 26,27-bisnor-25-hydroxyvitamin D3, and 22-27-hexanor-20-hydroxyvitamin D3 and the corresponding 5,6-trans isomers have been synthesized. All compounds were tested for their ability to induce intestinal calcium transport and bone calcium mobilization in normal and anephric rats. The 27-nor- and 26,27-bisnor-25-hydroxyvitamin D3 analog are capable of stimulating intestinal calcium transport and bone calcium mobilization in normal rats but are 10 to 100 times less active than 25-hydroxyvitamin D3. Although these analogs are inactive in anephric rats, their corresponding 5,6-trans isomer are capable of stimulating both intestine and bone activity in these animals. The 22-27-hexanor-20-hydroxyvitamin D3 and its corresponding 5,6-trans isomer are incapable of stimulating either intestinal calcium transport or bone calcium mobilization. These results suggest that minor alterations in the side chain significantly decrease the biopotency of 25-hydroxyvitamin D3. Since these analogs are biologically active in normal but not in anephric animals, it appears that the kidney 1alpha-hydroxylation is necessary for activity. Since 22-27-hexanor=20-hydroxyvitamin D3 and its corresponding 5,6-trans analog are biologically inactive, it is likely that at least part of the side chain is necessary for 25-hydroxyvitamin D3 to stimulate intestinal calcium transport and bone calcium mobilization.     

Differential effects of Vitamin D analogs on bone formation and resorption

Deficiency in Vitamin D and its metabolites leads to a failure in bone formation primarily caused by dysfunctional mineralization, suggesting that Vitamin D analogs might stimulate osteoblastic bone formation and mineralization. In this study, we compare the effect of selected Vitamin D analogs and active metabolite, 1alpha,25-dihydroxyvitamin D(3), 19-nor-1alpha, 25-dihydroxyvitamin D(2), and 1alpha-hydroxyvitamin D(2) or 1alpha,25-dihydroxyvitamin D(2) on bone formation and resorption. In a mouse calvariae bone primary organ culture system, all Vitamin D analogs and metabolite tested-stimulated collagen synthesis in a dose-dependent manner and 19-nor-1alpha, 25-dihydroxyvitamin D(2) was the most efficacious among three. 19-nor-1alpha, 25-dihydroxyvitamin D(2) and 1alpha,25-dihydroxyvitamin D(2) showed similar potencies and 1alpha,25-dihydroxyvitamin D(3) was less potent than others. Osteocalcin was also up-regulated in a dose-dependent manner, suggesting that the three Vitamin D analogs have the equal potencies on bone formation. 25-Hydroxyvitamin D-24-hydroxylase expression was induced in a dose-dependent manner and 19-nor-1alpha, 25-dihydroxyvitamin D(2) was less potent than other two compounds. In a mouse calvariae organ culture, all induced a net calcium release from calvariae in a dose-dependent manner, but the potency is in the order of 1alpha,25-dihydroxyvitamin D(2) congruent with1alpha,25-dihydroxyvitamin D(3)>19-nor-1alpha, 25-dihydroxyvitamin D(2). In a Vitamin D/calcium-restricted rat model, all caused an elevation in serum calcium in a dose-dependent manner. There is no significant difference between 1alpha,25-dihydroxyvitamin D(3) and 1alpha-hydroxyvitamin D(2) in potencies, but 19-nor-1alpha, 25-dihydroxyvitamin D(2) is at least 10-fold less potent than the other two compounds. Our results suggest that Vitamin D analogs have direct effects on bone resorption and formation, and 19-nor-1alpha, 25-dihydroxyvitamin D(2) may be more effective than 1alpha,25-dihydroxyvitamin D(3) and 1alpha-hydroxyvitamin D(2) on stimulating anabolic bone formation.     

Side-chain oxidation of vitamin D3 in mouse kidney mitochondria: effect of the Hyp mutation and 1,25-dihydroxyvitamin D3 treatment

Side-chain oxidation of vitamin D is an important degradative pathway. In the present study we compared the enzymes involved in side-chain oxidation in normal and Hyp mouse kidney. Homogenates of normal mouse kidney catalyze the conversion of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3, 24-oxo-25-hydroxyvitamin D3 and 24-oxo-23,25-dihydroxyvitamin D3. After subcellular fractionation, total side-chain oxidative activity, estimated by the sum of the three products synthesized per milligram protein under initial rate conditions, coincided with the mitochondrial enzyme marker succinate-cytochrome-c reductase. Treatment of normal mice with 1,25-dihydroxyvitamin D3 (1.5 ng/g) resulted in an eightfold increase in mitochondrial enzyme activity, with no change in apparent Km but a significant rise in Vmax. With 24,25-dihydroxyvitamin D3 as the substrate, normal renal mitochondria produced 24-oxo-25-hydroxyvitamin D3 and 24-oxo-23,25-dihydroxyvitamin D3, and the synthesis of these metabolites could be increased sixfold by pretreatment with 1,25-dihydroxyvitamin D3. In the Hyp mouse, the side-chain oxidation pathway showed similar subcellular distribution of enzyme activity. However, product formation from 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 was twofold greater in mutant than in normal mitochondria. Furthermore, 1,25-dihydroxyvitamin D3 pretreatment of Hyp mice resulted in a 3.4-fold increase over basal metabolism of both 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3. These results demonstrate that (i) kidneys from normal and Hyp mice possess basal and 1,25-dihydroxyvitamin D3 inducible enzyme system(s) in the mitochondrial fraction, which catalyze the side-chain oxidation of 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3, and (ii) the Hyp mutation appears to perturb the renal metabolism of both substrates only in the basal state.     

Rodent macrophages metabolize 25-hydroxyvitamin D3in,vitro

Rodent macrophages metabolized 25-hydroxyvitamin D₃ to an unidentified metabolite during $\text{in}\text{,}\text{vitro}$ incubations. The production of this macrophage-derived metabolite of 25-hydroxyvitamin D₃ increased as the substrate concentration was raised. A two step high pressure liquid chromatography system revealed a unique elution position of this macrophage-derived metabolite that did not match the elution positions of any of the vitamin D₃ metabolites available in this laboratory. This unique metabolite was formed $\text{in}\text{,}\text{vitro}$ within one minute by incubated macrophages although its formation increased gradually up to 60 minutes of incubation.     

Evaluation of C-2-substituted 19-nor-1alpha,25-dihydroxyvitamin D3 analogs as therapeutic agents for prostate cancer

1alpha,25-Dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) is known to inhibit the proliferation and invasiveness of prostate cancer cells. However, 1alpha,25(OH)(2)D(3) can cause hypercalcemia and is not suitable as a therapeutic agent. 19-Nor-vitamin D derivatives are known to be less calcemic when administered systemically. In order to develop more potent anti-cancer agents with less calcemic side effect, we therefore utilized (3)H-thymidine incorporation as an index for cell proliferation and examined the antiproliferative activities of nine C-2-substituted 19-nor-1alpha,25(OH)(2)D(3) analogs in the immortalized PZ-HPV-7 normal prostate cell line. Among the nine analogs we observed that the substitution with 2alpha- or 2beta-hydroxypropyl group produced two analogs having antiproliferative potency that is approximately 500- to 1000-fold higher than 1alpha,25(OH)(2)D(3). The (3)H-thymidine incorporation data were supported by the cell counting data after cells were treated with 1alpha,25(OH)(2)D(3), 19-nor-2alpha-(3-hydroxypropyl)-1alpha,25(OH)(2)D(3) or 19-nor-2beta-(3-hydroxypropyl)-1alpha,25(OH)(2)D(3) for 7 days. 19-Nor-2alpha-(3-hydroxypropyl)-1alpha,25(OH)(2)D(3) and 19-nor-2beta-(3-hydroxypropyl)-1alpha,25(OH)(2)D(3) were also shown to be about 10-fold more active than 1alpha,25(OH)(2)D(3) in cell invasion studies using prostate cancer cells. In conclusion, a substitution at the C-2 position of 19-nor-1alpha,25(OH)(2)D(3) molecule with a hydroxypropyl group greatly increased the antiproliferative and anti-invasion potencies. Thus, these two analogs could be developed to be effective therapeutic agents for treating early and late stages of prostate cancer.     

Fluorometric assay of 25-hydroxyvitamin D3 and 24R,25-dihydroxyvitamin D3 in plasma.

The first practical fluorometric assay of plasma 25-hydroxyvitamin D3 (25-OH-D3) and 24R,25-dihydroxyvitamin D3 (24,25-(OH)2D3) is described. The method uses a highly fluorescent dienophile, 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1, 2,4- triazoline-3,5-dione (DMEQ-TAD), to fluorescence-label vitamin D. Vitamin D metabolites were roughly purified with a short cartridge column followed by HPLC, labeled with DMEQ-TAD, and the product was analyzed on HPLC. In the assay of 25-OH-D3 the new fluorometric method was compared with the HPLC-uv method and was confirmed to be as accurate and reliable (CV, 4-5%) as the HPLC-uv method. Plasma 24,25-(OH)2D3 was accurately assayed by the HPLC-FL method, where the standard addition method was successfully used to calculate the overall recovery.