Enzymatic properties of mouse 25-hydroxyvitamin D3 1 alpha-hydroxylase expressed in Escherichia coli.

Renal 25-hydroxyvitamin D3 1 alpha-hydroxylase cDNA cloned from the kidneys of mice lacking the vitamin D receptor was expressed in Escherichia coli JM109. As expected, the bacterially-expressed enzyme catalyzes the 1 alpha-hydroxylation of 25-hydroxyvitamin D3 with a Michaelis constant, K(m), value of 2.7 microM. Unexpectedly, the enzyme also hydroxylates the 1 alpha-position of 24,25-dihydroxyvitamin D3 with a K(m) of 1.3 microM, and a fourfold higher Vmax/K(m) compared with the 25-hydroxyvitamin D3 hydroxylase activity, suggesting that 24,25-dihydroxyvitamin D3 is a better substrate than 25-hydroxyvitamin D3 for 1 alpha-hydroxylase. In addition, the enzyme showed 1 alpha-hydroxylase activity toward 24-oxo-25-hydroxyvitamin D3. However, it showed only slight activity towards 23,25-dihydroxyvitamin D3 and 24-oxo-23,25-dihydroxyvitamin D3, and no detectable activity towards vitamin D3 and 24,25,26,27-tetranor-23-hydroxyvitamin D3. These results suggest that the 25-hydroxyl group of vitamin D3 is essential for the 1 alpha-hydroxylase activity and the 24-hydroxyl group enhances the activity, but the 23-hydroxyl group greatly reduced the activity. Another remarkable finding is that living recombinant E. coli cells can convert the substrates into the 1 alpha-hydroxylated products, suggesting the presence of a redox partner of 1 alpha-hydroxylase in E. coli cells.     

Metabolism of 25-hydroxyvitamin D3 in renal slices from the X-linked hypophosphatemic (Hyp) mouse: abnormal response to fall in serum calcium

The effect of the X-linked Hyp mutation on 25-hydroxyvitamin D3 (25-OH-D3) metabolism in mouse renal cortical slices was investigated. Vitamin D replete normal mice and Hyp littermates fed the control diet synthesized primarily 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3); only minimal synthesis of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) was detected in both genotypes and 1,25-(OH)2D3 formation was not significantly greater in Hyp mice relative to normal littermates, despite hypophosphatemia and hypocalcemia in the mutants. Calcium-deficient diet fed to normal mice reduced serum calcium (p less than 0.01), increased renal 25-hydroxyvitamin D3-1-hydroxylase (1-OHase) activity (p less than 0.05), and decreased 25-hydroxyvitamin D3-24-hydroxylase (24-OHase) activity (p less than 0.05). In contrast, Hyp littermates on the calcium-deficient diet had decreased serum calcium (p less than 0.01), without significant changes in the renal metabolism of 25-OH-D3. Both normal and Hyp mice responded to the vitamin D-deficient diet with a fall in serum calcium (p less than 0.01), significantly increased renal 1-OHase, and significantly decreased renal 24-OHase activities. In Hyp mice, the fall in serum calcium on the vitamin D-deficient diet was significantly greater than that observed on the calcium-deficient diet. Therefore the ability of Hyp mice to increase renal 1-OHase activity when fed the vitamin D-deficient diet and their failure to do so on the calcium-deficient diet may be related to the resulting degree of hypocalcemia. The results suggest that although Hyp mice can respond to a disturbance of calcium homeostasis, the in vivo signal for the stimulation of renal 1-OHase activity may be set at a different threshold in the Hyp mouse; i.e. a lower serum calcium concentration is necessary for Hyp mice to initiate increased synthesis of 1,25(-OH)2D3.     

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)).     

23-Keto-25-hydroxyvitamin D3: a vitamin D3 metabolite with high affinity for the 1,25-dihydroxyvitamin D specific cytosol receptor

A new metabolite of 23,25-dihydroxyvitamin D3 has been generated with kidney homogenates prepared from vitamin D treated chicks. The metabolite was purified with three high-performance liquid chromatographic steps and was identified as 23-keto-25-hydroxyvitamin D3 by ultraviolet absorption spectroscopy, mass spectrometry, and chemical reactivity. The R stereoisomer of 23,25-dihydroxyvitamin D3 was 10-fold more effective as an in vitro precursor to 23-keto-25-hydroxyvitamin D3 than was the naturally occurring S stereoisomer. Approximately 500 ng of 23-keto-25-hydroxyvitamin D3 was necessary to produce the same degree of intestinal-calcium transport as 25 ng of vitamin D3--a difference of about 20-fold. 23-Keto-25-hydroxyvitamin D3 was not active at stimulating bone calcium resorption at the doses and times tested. This new vitamin D3 metabolite, however, had greater affinity than 25-hydroxyvitamin D3 to both the rat plasma vitamin D binding protein and the 1,25-dihydroxyvitamin D specific cytosol receptor. Heretofore, only 1 alpha-hydroxylated metabolites of 25-hydroxyvitamin D3 or analogues possessing a pseudo 1 alpha-hydroxy group were known to bind to the 1,25-dihydroxyvitamin D receptor with higher affinity than 25-hydroxyvitamin D3. Ketone formation at the 23 position, therefore, is the first side-chain modification of 25-hydroxyvitamin D3 that results in enhanced binding to the 1,25-dihydroxyvitamin D receptor binding protein.     

Kinetic properties of 25-hydroxyvitamin D- and 1,25-dihydroxyvitamin D-24-hydroxylase from chick kidney

1,25-Dihydroxyvitamin D3 induces both 25-hydroxyvitamin D3- and 1,25-dihydroxyvitamin D3- 24-hydroxylase activities. However, whether 24-hydroxylation of these substrates is catalyzed by a single enzyme is unknown. We have examined the substrate specificity of the enzyme using the solubilized and reconstituted chick renal mitochondrial 24-hydroxylase enzyme system. The soluble enzyme catalyzes 24-hydroxylation of both substrates. The apparent Km of the 24-hydroxylase for 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 were 1.47 and 0.14 microM, respectively. Kinetic studies demonstrated that 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 act as competitive inhibitors with respect to each other. 1,25-Dihydroxyvitamin D3 inhibited the production of 24,25-dihydroxyvitamin D3 with an apparent Ki of 0.09 microM and 25-hydroxyvitamin D3 inhibited the production of 1,24,25-trihydroxyvitamin D3 with an apparent Ki of 3.9 microM. These results indicate that chick 24-hydroxylase preferentially hydroxylates 1,25-dihydroxyvitamin D3 and support the idea that the 24-hydroxylation of these substrates is catalyzed by a single enzyme.     

Assay and properties of 25-hydroxyvitamin D3 23-hydroxylase. Evidence that 23,25-dihydroxyvitamin D3 is a major metabolite in 1,25-dihydroxyvitamin D3-treated or fasted guinea pigs.

Incubation of 25-hydroxyvitamin D3 with kidney cortex mitochondria from 1,25-dihydroxyvitamin D3-treated guinea pigs resulted in the formation of 23,25-dihydroxyvitamin D3 as the major product. The identity of the product was verified by g.c.-m.s. and quantification was performed by h.p.l.c. The rates of the reaction were in the range 1.0-1.8 pmol/min per mg of mitochondrial protein (at 37 degrees C), which were 5-10 times the rates of formation of 24,25-dihydroxyvitamin D3. In mitochondrial preparations from untreated guinea pigs, the rate of 23-hydroxylation was below detection limit (0.02 pmol/min per mg of mitochondrial protein). Fasting the animals for 24 h induced the 23-hydroxylase almost as efficiently as treatment with 1,25-dihydroxyvitamin D3, with a concomitant depression of the 1 alpha-hydroxylase. The 23-hydroxylase reaction required oxidizable substrate, was decreased by low O2 partial pressures and inhibited by CO or the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. It was stimulated by the respiratory-chain inhibitors rotenone, antimycin A and KCN. These results indicate that the guinea-pig renal mitochondrial 23-hydroxylase is a cytochrome P-450 and that the reducing equivalents are primarily supplied by NADPH via the energy-dependent transhydrogenase.     

End product inhibition of hepatic 25-hydroxyvitamin D production in the rat: specificity and kinetics

The role of vitamin D metabolites in the regulation of hepatic 25-hydroxyvitamin D production was investigated by examining the effects of 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, and 24,25-dihydroxyvitamin D on the synthesis of [25-3H]hydroxyvitamin D by rachitic rat liver homogenates. Production of [25-3H]hydroxyvitamin D was inhibited by 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, but not by 24,25-dihydroxyvitamin D. 25-Hydroxyvitamin D increased the Km of the vitamin D-25-hydroxylase enzyme(s), while 1,25-dihydroxyvitamin D decreased the Vmax with a Ki of 88.7 ng/ml. Inhibition of hepatic 25-hydroxyvitamin D production by 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D may be another control mechanism to regulate circulating vitamin D levels.     

Metabolism of 25-hydroxyvitamin D3 by microsomal and mitochondrial vitamin D3 25-hydroxylases (CYP2D25 and CYP27A1): a novel reaction by CYP27A1

The metabolism of 25-hydroxyvitamin D(3) was studied with a crude mitochondrial cytochrome P450 extract from pig kidney and with recombinant human CYP27A1 (mitochondrial vitamin D(3) 25-hydroxylase) and porcine CYP2D25 (microsomal vitamin D(3) 25-hydroxylase). The kidney mitochondrial cytochrome P450 catalyzed the formation of 1alpha,25-dihydroxyvitamin D(3), 24,25-dihydroxyvitamin D(3) and 25,27-dihydroxyvitamin D(3). An additional metabolite that was separated from the other hydroxylated products on HPLC was also formed. The formation of this 25-hydroxyvitamin D(3) metabolite was dependent on NADPH and the mitochondrial electron transferring protein components. A monoclonal antibody directed against purified pig liver CYP27A1 immunoprecipitated the 1alpha- and 27-hydroxylase activities towards 25-hydroxyvitamin D(3) as well as the formation of the unknown metabolite. These results together with substrate inhibition experiments indicate that CYP27A1 is responsible for the formation of the unknown 25-hydroxyvitamin D(3) metabolite in kidney. Recombinant human CYP27A1 was found to convert 25-hydroxyvitamin D(3) into 1alpha,25-dihydroxyvitamin D(3), 25,27-dihydroxyvitamin D(3) and a major metabolite with the same retention time on HPLC as that formed by kidney mitochondrial cytochrome P450. Gas chromatography-mass spectrometry (GC-MS) analysis of the unknown enzymatic product revealed it to be a triol different from other known hydroxylated 25-hydroxyvitamin D(3) metabolites such as 1alpha,25-, 23,25-, 24,25-, 25,26- or 25,27-dihydroxyvitamin D(3). The product had the mass spectrometic properties expected for 4beta,25-dihydroxyvitamin D(3). Recombinant porcine CYP2D25 converted 25-hydroxyvitamin D(3) into 1alpha,25-dihydroxyvitamin D(3) and 25,26-dihydroxyvitamin D(3). It can be concluded that both CYP27A1 and CYP2D25 are able to carry out multiple hydroxylations of 25-hydroxyvitamin D(3).     

Support of embryonic chick survival by vitamin D metabolites

The provision of 1,25-dihydroxyvitamin D3 as the only source of dietary vitamin D3 to laying hens failed to support normal embryonic development in their fertile eggs. Significant (P less than .001) improvement in embryonic survival to hatching in these eggs resulted from injections of 1,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin D3, 25-hydroxyvitamin D3, or 24,24-difluoro-25-hydroxyvitamin D3 prior to incubation. Maximum embryonic survival with lowest embryonic mortality was observed when 0.20 micrograms/egg of 1,25-dihydroxyvitamin D3 or 0.60 micrograms/egg 25-hydroxyvitamin D3 was injected. These results indicate that several forms of vitamin D, two of which cannot be converted to 24,25-dihydroxyvitamin D3, can provide this activity; and of the vitamin D compounds tested, 1,25-dihydroxyvitamin D3 may be the most active in supporting embryonic survival in the chick when delivered directly by injection.     

The mechanism of 1,25-dihydroxyvitamin D(3) autoregulation in keratinocytes

The synthesis of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) from its precursor, 25-dihydroxyvitamin D(3) (25(OH)D(3)), is catalyzed by the mitochondrial cytochrome P450 enzyme 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-hydroxylase). It has been generally assumed that 1,25(OH)(2)D(3) inhibits the activity of this enzyme by regulating its expression at the genomic level. We confirmed that 1,25(OH)(2)D(3) reduced the apparent conversion of 25(OH)D(3) to 1,25(OH)(2)D(3) while stimulating the conversion of 1,25(OH)(2)D(3) and 25(OH)D(3) to 1,24,25(OH)(3)D(3) and 24,25(OH)(2)D(3), respectively. However, 1,25(OH)(2)D(3) failed to reduce the abundance of its mRNA or its encoded protein in human keratinocytes. Instead, when catabolism of 1,25(OH)(2)D(3) was blocked with a specific inhibitor of the 25-hydroxyvitamin D(3)-24-hydroxylase (24-hydroxylase) all apparent inhibition of 1alpha-hydroxylase activity by 1,25(OH)(2)D(3) was reversed. Thus, the apparent reduction in 1alpha-hydroxylase activity induced by 1,25(OH)(2)D(3) is due to increased catabolism of both substrate and product by the 24-hydroxylase. We believe this to be a unique mechanism for autoregulation of steroid hormone synthesis.     

Metabolism and binding properties of 24,24-difluoro-25-hydroxyvitamin D3

To evaluate possible functional roles for 24,25-dihydroxyvitamin D₃, 24,24-difluoro-25-hydroxyvitamin D₃ has been synthesized and shown to be equally as active as 25-hydroxyvitamin D₃ in all known functions of vitamin D. The use of the difluoro compound for this purpose is based on the assumption that the C-F bonds are stable in vivo and that the fluorine atom does not act as hydroxyl in biological systems. No 24,25-dihydroxyvitamin D₃ was detected in the serum obtained from vitamin D-deficient rats that had been given 24,24-difluoro-25-hydroxyvitamin D₃, while large amounts were found when 25-hydroxyvitamin D₃ was given. Incubation of the 24,24-difluoro compound with kidney homogenate prepared from vitamin D-replete chickens failed to produce 24,25-dihydroxyvitamin D₃, while the same preparations produced large amounts of 24,25-dihydroxyvitamin D₃ from 25-hydroxyvitamin D₃. Kidney homogenate prepared from vitamin D-deficient chickens produced 24,24-difluoro-1,25-dihydroxyvitamin D₃ from 24,24-difluoro-25-hydroxyvitamin D₃ and 1,25-dihydroxyvitamin D₃ from 25-hydroxyvitamin D₃. In binding to the plasma transport protein for vitamin D compounds, 24,24-difluoro-25-hydroxyvitamin D₃ is less active than 25-hydroxyvitamin D₃ and 24R,25-dihydroxyvitamin D₃. In binding to the chick intestinal cytosol receptor, 24,24-difluoro-25-hydroxyvitamin D₃ is more active than 25-hydroxyvitamin D₃ which is itself more active than 24R,25-dihydroxyvitamin D₃. The 24,24-difluoro-1,25-dihydroxyvitamin D₃ is equal to 1,25-dihydroxyvitamin D₃, and both are 10 times more active than 1,24R,25-trihydroxyvitamin D₃ in this system. These results provide strong evidence that the C-24 carbon of 24,24-difluoro-25-hydroxyvitamin D₃ cannot be hydroxylated in vivo, and, further, the 24-F substitution acts similar to H and not to OH in discriminating binding systems for vitamin D compounds.     

25-hydroxy-vitamin d metabolism in human colon cancer cells during tumor progression

RT-PCR analysis showed elevated expression of 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-OHase) and of 25-hydroxyvitamin D-24-hydroxylase (24-OHase) in well differentiated human colon carcinomas in comparison to normal mucosa. Further tumor progression is associated with a rise in 1alpha-OHase but with no significant change in 24-OHase mRNA expression. Accordingly, HPLC analysis of 25-hydroxy-vitamin D3 metabolism in freshly isolated tumor cells indicated that well to moderately differentiated colon cancers in situ are able to produce 1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3) and convert it through 24-OHase activity into side-chain modified metabolites, 1,24,25-(OH)3-D3 and 1,25-(OH)2- 24-oxo-D3. Likewise, 25-(OH)-D3 is metabolized into 24,25-(OH)2D3, 23,25-(OH)2D3, and 23,25-(OH)2-24-oxo-D3. Poorly-differentiated cancers expressed low levels of 1alpha-OHase mRNA, whereas 24-OHase was even over-expressed. RT-PCR and HPLC analysis of vitamin D metabolism in primary culture cell clones strongly suggested that the extent of endogenously produced 1alpha,25-(OH)2-D3 was inversely related to 24-OHase activity, which could thus limit the antimitotic efficacy of 1alpha,25-(OH)2-D3 particularly at late stages of colon cancer progression.     

Identification of 24,25,26,27-tetranor-23-hydroxyvitamin D3 as a product of the renal metabolism of 24,25-dihydroxyvitamin D3

By cochromatography, mass spectrometry, and chemical derivatization, we have shown that a metabolite isolated from the perfused rat kidney incubated with 24-(R),25-dihydroxyvitamin D3 is indistinguishable from chemically synthesized 24,25,26,27-tetranor-23-hydroxyvitamin D3. The new metabolite is also produced from 24-oxo-25-hydroxyvitamin D3 but not from 23(S),25-dihydroxyvitamin D3. Enzymes required for the synthesis of the new metabolite are absent in the vitamin D deplete animal but are induced along with the 25-hydroxyvitamin-D3 24-hydroxylase by vitamin D repletion. The pathway of 24,25-dihydroxyvitamin D3 metabolism in the perfused kidney is stimulated by pre-treatment of the rat with large doses of vitamin D3, suggesting that the pathway is a degradative one.     

Effects of 24,25(OH)2D3, 1,25(OH)2D3 and 25(OH)D3 on alkaline and tartrate-resistant acid phosphatase activities in fetal rat calvaria

The aim of this work was to evaluate the effects of 24,25-dihydroxyvitamin D3, 24,25(OH)2D3, on alkaline phosphatase (AP) and tartrate-resistant acid phosphatase (TRAP) activities in fetal rat calvaria cultures. These actions were compared with those of 1,25-dihydroxyvitamin D3, 1,25(OH)2D3, and 25-hydroxyvitamin D3, 25(OH)D3, in similar experimental conditions. At 10 min, 30 min and at 24 h incubation time, 1,25(OH)2D3 (10(-10)M) and 25(OH)D3 (10(-7) M) produced a significant increase in AP and TRAP activities compared to control group (without vitamin D metabolites). However, 24,25(OH)2D3 (10(-7) M) only produced effects on phosphatase activities similar to those produced by 1,25(OH)2D3 and 25(OH)D3, after 24 h incubation time. These findings suggest that 1,25(OH)2D3 and 25(OH)2D3 could carry out actions in minutes (nongenomic mechanism), while 24,25(OH)2D3 needs longer periods of time to perform its biological actions (genomic mechanism).     

Induction of 25-OH-vitamin D3 24- and 23-hydroxylase activities in partially purified renal extracts from pigs given exogenous 1,25-(OH)2D3

A renal mitochondrial cytochrome P 450 preparation from pigs treated with exogenous 1,25-(OH)2D3 was reconstituted with an NADPH-generating system, adrenodoxin and adrenodoxin reductase. The reconstituted system catalyzed the conversion of the substrate, 25-OH-D3, to metabolites comigrating with authentic 23,25-(OH)2D3 and 24,25-(OH)2D3 in both straight- and reverse-phase high-performance liquid chromatography systems, which achieve separation of these metabolites from each other as well as from other vitamin D metabolites. The putative 23,25-(OH)2D3 product was resistant to periodate treatment, while the 24,25-(OH)2D3 product was sensitive, providing additional evidence for the identity of the products. Although induction of 24-hydroxylase activity has been studied using renal homogenates from several species, only recently have techniques become available to study the activity of the enzyme in a solubilized and reconstituted form. Using these techniques, the present study shows that production of 24,25-(OH)2D3 was increased more than 80-fold with 1,25-(OH)2D3 treatment compared with untreated controls, an effect much greater than that previously observed with homogenates. In addition, production of both 23,25-(OH)2D3 and 24,25-(OH)2D3 varied with substrate concentration and was consistent with a monooxygenase-linked enzyme reaction.     

Solubilization and reconstitution of chick renal mitochondrial 25-hydroxyvitamin D3 24-hydroxylase

Chick kidney mitochondrial 25-hydroxyvitamin D3 24-hydroxylase has been solubilized with sodium cholate and reconstituted with NADPH, beef adrenal ferredoxin, and beef adrenal ferredoxin reductase, each component being essential for maximal 24-hydroxylase activity. The product 24(R),25-dihydroxyvitamin D3 was identified by cochromatography with synthetic compound on straight-phase and reversed-phase high-performance liquid chromatography and by periodate oxidation. The enzyme has an apparent Km for 25-hydroxyvitamin D3 of 0.67 microM. At 1 microM 25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D3 production is linear with time for up to 15 min and with protein concentrations of up to 2 mg/mL. The antioxidant diphenyl-p-phenylenediamine (1.3 X 10(-4) M) has no effect on this reaction. Reconstituted 24-hydroxylase activity is enhanced by the addition of NaCl and KCl up to 100 mM, with higher concentrations having an inhibitory effect. 1 alpha-Hydroxylase is not present in this preparation from vitamin D replete chicks. The similarities of this reconstituted system to the 25-hydroxyvitamin D3 1 alpha-hydroxylase and the adrenal systems suggest that the 25-hydroxyvitamin D3 24-hydroxylase is also a cytochrome P-450 type mixed-function oxidase.     

Isolation and identification of 23,25-dihydroxyvitamin D3, an in vivo metabolite of vitamin D3

Vitamin D supplemented rats produce a metabolite of 25-hydroxy[3 alpha-3H]vitamin D3 that is easily separated from known metabolites by using high-performance liquid chromatography. The production of this metabolite in vivo as well as 1,25-dihydroxyvitamin D3, 24(R),25-dihydroxyvitamin D3, and 25-hydroxyvitamin D3 26,23-lactone is largely if not totally eliminated by nephrectomy. Kidney homogenates from vitamin D supplemented chickens incubated with 25-hydroxyvitamin D3 produce significant quantities of the new, unknown metabolite. This metabolite was isolated in pure form from such incubation mixtures by using both straight-phase and reversed-phase high-performance liquid chromatography. This metabolite has been positively identified as 23,25-dihydroxyvitamin D3 by ultraviolet absorption spectrophotometry, mass spectrometry, and derivatization. This structure was confirmed by chemical synthesis of both C-23 stereoisomers. Although the natural product exactly comigrates with one of the synthetic isomers, the exact stereochemistry of the natural product remains unknown. It is possible that this new metabolite is an intermediate in the biosynthesis of 25-hydroxyvitamin D3 26,23-lactone.     

Plasma levels of vitamin D metabolites in fetal and pregnant ewes

The plasma concentrations of calcium; inorganic phosphorus; 25-hydroxyvitamin D; 24,25-dihydroxyvitamin D; and 1,25-dihydroxyvitamin D were determined in sheep maternal and fetal arterial circulations. In addition, plasma concentrations of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D were determined simultaneously across the uterine and umbilical circulations. Fetal arterial levels of calcium (r = 0.560); inorganic phosphorus (r = -0.095); and 1,25-dihydroxyvitamin D (r = 0.040) were significantly higher than and did not correlate with maternal arterial levels. Maternal levels of 25-hydroxyvitamin D were significantly higher than and correlated (r = 0.693) with fetal 25-hydroxyvitamin D levels. No significant difference existed between maternal and fetal arterial levels of 24,25-dihydroxyvitamin D. No significant difference was detected in the concentrations of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D across the uterine or umbilical circulations.     

Characterization of new conjugated metabolites in bile of rats administered 24,25-dihydroxyvitamin D(3) and 25-hydroxyvitamin D(3)

The characterization of new conjugated vitamin D metabolites in rat bile was performed using HPLC, liquid chromatography/tandem mass spectrometry combined derivatization, and GC-MS. After the administration of 24,25-dihydroxyvitamin D(3) to rats, 23, 25-dihydroxy-24-oxovitamin D(3) 23-glucuronide, 3-epi-24, 25-dihydroxyvitamin D(3) 24-glucuronide, and 24,25-dihydroxyvitamin D(3) 3-sulfate were obtained as new biliary metabolites together with 24,25-dihydroxyvitamin D(3) 3- and 24-glucuronides. The above metabolites, except 24,25-dihydroxyvitamin D(3) 3-glucuronide, were obtained from rats dosed with 25-hydroxyvitamin D(3). 23, 25-Dihydroxyvitamin D(3) 23-glucuronide was also obtained from the bile of rats administered 25-hydroxyvitamin D(3) in addition to its 3-glucuronide, 25-glucuronide, and 3-sulfate. Thus, it was found that 24,25-dihydroxyvitamin D(3) and 25-hydroxyvitamin D(3) were directly conjugated as glucuronide and sulfate, whereas at the C-23 position, they were hydroxylated and then conjugated. Furthermore, we found that the C-3 epimerization acts as one of the important pathways in vitamin D metabolism.     

Metabolism and pharmacokinetics of 24,25-dihydroxyvitamin D3 in the vitamin D3-replete rat

The time course of in vivo metabolism of 24,25-dihydroxyvitamin D3 in rats has been examined. Several tissues were surveyed in an effort to discover new metabolites of 24,25-dihydroxyvitamin D3 and to estimate the concentrations of previously identified metabolites. Rapidly growing male rats were dosed with 24,25-dihydroxyvitamin D3 orally until plasma concentrations of 24,25-dihydroxyvitamin D3 were at steady state. 24,25-Dihydroxyvitamin [3-3H]D3 was then administered. At 10 min and 1, 6, 15, 24, 96, and 192 h after dosing, the animals were killed, and plasma, liver, intestine, and bones were analyzed with a newly developed gradient straight-phase high performance liquid chromatography system. The high performance liquid chromatography system is capable of base-line resolution of most of the major vitamin D metabolites. 24,25-Dihydroxyvitamin D3 clearance from plasma, liver, and kidney but not intestine followed a two-compartment model. 24,25-Dihydroxyvitamin D3 disappeared from plasma with a half-life of 0.55 h (fast phase) and 73.8 h (slow phase). Only two lipid-soluble metabolites of 24,25-dihydroxyvitamin D3 were detected: 24-oxo-25-hydroxyvitamin D3 and 1,24,25-trihydroxyvitamin D3. These compounds circulate at very low concentrations in the plasma (50 pg/ml of plasma).