1,25-Dihydroxyvitamin D3 receptor in bovine thymus gland

1,25-Dihydroxyvitamin D₃ [1,25-(OH)₂D₃] receptor was characterized after partial purification of thymus cytosol by ammonium sulfate fractionation. The 1,25-(OH)₂D₃ receptor sediments at 3.7S in 5–20% sucrose gradients. The binding of 1,25-(OH)₂D₃ in thymic cytosol was a saturable process with high affinity (Kd = 0.12?0.48 nM) at 4°C. Competition for 1,25-(OH)₂[³H]D₃ receptor by nonradioactive analogs demonstrated the affinities of these analogs to be in order; 1,25-(OH)₂D₃ = 1,24R,25-(OH)₃D₃ = 1,25S,26-(OH)₃D₃ = 1,25R,26-(OH)₃D₃ > 1,25-(OH)₂D₃-26,23 lactone > 25-OHD₃ > 23R,25-(OH)₂D₃ > 24R,25-(OH)₂D₃ > 23S,25-(OH)₂D₃ ? 25-OHD₃-26,23 lactone. The receptor bound to DNA cellulose columns in low salt buffer and eluted as a single peak at 0.21 M KCl. These findings provide evidence that the thymus possesses a 1,25-(OH)₂D₃ receptor with properties indistinguishable from 1,25-(OH)₂D₃ receptors in other tissues.     

25-OHD3-26,23-lactone: a metabolite of vitamin D3 that is 5 times more potent than 25-OHD3 in the rat plasma competitive protein binding radioassay.

A new metabolite of Vitamin D₃ (25-OHD₃-26,23-lactone) has been found in the plasma of Vitamin D₃-toxic pigs and cows. This metabolite is at least 5 times more potent than 25-OHD₃ in the displacement of [³H]-25-OHD₃ from rat plasma protein binding sites under short-term incubation. This metabolite co-migrates with 24,25-(OH)₂D₃ on Sephadex LH-20 columns developed in chloroform:hexane 65:35 and with 25,26-(OH)₂D₃ on Sephadex LH-20 columns developed in hexane:chloroform:methanol 9:1:1. The presence of 25-OHD₃-26,23-lactone represents a possible contaiminant in the assay of 24,25-(OH)₂D₃ or 25,26-(OH)₂D₃ if only Sephadex LH-20 is used for pre-assay purification. 25-OHD₃-26,23-lactone is, however, resolved from 24,25-(OH)₂D₃ by high pressure liquid chromatography (HPLC) using Zorbax Sil silicic acid columns developed in either isopropanol:hexane 8:92 or isopropanol:methylene chloride 2.5:96.5. We assayed for the presence of this new metabolite of Vitamin D₃ and found it to be present in normal pig plasma and undetectable in normal cow plasma. Concentrations were elevated to 10–20 ng/ml following massive injection of Vitamin D₃ to both species.     

Vitamin D metabolite-binding proteins in human tissue

Serum and post-microsomal supernatants of human lymphocyte, erythrocyte, skeletal muscle and parathyroid adenoma homogenates were examined for specific binding of 25-hydroxycholecalciferol (25-OHD₃) and 1,25-dihydroxycholecalciferol (1,25-(OH)₂D₃). Muscle, lymphocytes and parathyroid adenomata extracts contained a 6-S 25-OHD₃-binding protein which was not found in erythrocyte extracts, and which was distinct from the smaller serum transport α-globulin. A cathodal, 1,25-(OH)₂D₃-binding protein, which sedimented at 3–4 S was also detected in parathyroid tissue. These observations suggest the possibility of direct physiologic interaction between vitamin D metabolites and nucleated human tissues other than intestine and bone.     

Quantitation of vitamin D and its metabolites and their plasma concentrations in five species of animals

Chromatographic methods suitable for the resolution of 24,25-dihydroxyvitamin D₃, 24,25-dihydroxyvitamin D₂, 25-hydroxyvitamin D₃-26,23 lactone, and 25,26-dihydroxyvitamin D₂ are described. These four metabolites comigrated in high-pressure liquid chromatography on silicic acid columns developed in 11:89 isopropanol:hexane. Adequate resolution was achieved by subjecting the four-metabolite complex to high-pressure liquid chromatography column developed in 2:98 isopropanol:methylene chloride. This additional chromatographic step, coupled with modifications of assay procedures previously described, allowed for the estimation of plasma concentrations of vitamin D₂, vitamin D₃, 25-hydroxyvitamin D₂, 25-hydroxyvitamin D₃, 24,25-dihydroxyvitamin D₂, 24,25-dihydroxyvitamin D₃, 25,26 dihydroxyvitamin D₂, 25,26-dihydroxyvitamin D₃, 25-hydroxyvitamin D₃-26,23 lactone, and 1,25-dihydroxyvitamin D (1,25-dihydroxyvitamin D₂ plus 1,25-dihydroxyvitamin D₃). The samples automatically were introduced onto the high-pressure liquid chromatography columns with a Waters 710A “intelligent” processor. The metabolites were automatically collected with the aid of a programmable timer that advanced a fraction collector at predetermined intervals. The assays were used to determine the plasma vitamin D and vitamin D metabolite concentrations in five species of adult farm animals.     

Influence of ultraviolet B radiation on vitamin D3 metabolism in vitamin D3-resistant New World primates

We investigated the occurrence of rickets in adolescent tamarins (Saguinus imperator) residing at the Los Angeles Zoo. Compared to tamarins in the same colony without clinical evidence of bone disease (N = 6), rachitic platyrrhines (N = 3) had a decrease in their serum calcium concentration (P < .05). The affected tamarins also had lower serum 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) levels than did nonaffected colony mates, but 2–10-fold higher concentrations than in Old World primates of a comparable developmental stage. New World primates in many different genera are known to exhibit target organ resistance to the active vitamin D₃ metabolite, 1,25-(OH)₂D₃, compensated by maintenance of high circulating concentrations of 1,25-(OH)₂D₃. The relatively low serum 1,25-(OH)₂D₃ concentration in rachitic tamarins and ultraviolet B radiation deficient environment of these primates suggested that bone disease may be linked to a deficiency in substrate for 1,25-(OH)₂D₃, 25 hydroxyvtamin D₃ (25-OHD₃). Chronic exposure of platyrrhines in three different vitamin D resistant genera to an artificial UVB source resulted in 1) a significant increase in the mean serum 25-OHD₃ (P < .001) and 1,25-(OH)₂D₃ (P < .02) level over that encountered in platyrrhines not exposed to UVB; and 2) prevention of rachitic bone disease in irradiated individuals. These data further show that the serum 25-OHD₃ and 1,25-OH₂D₃ levels are positively correlated in vitamin D-resistant platyrrhines (r = 0.64; P= .0014) and suggest that a compromise in cutaneous vitamin D₃ production by means of UVB deprivation may limit necessary 1,25-(OH)₂D₃ production. © 1992 Wiley-Liss, Inc.     

Synthesis and function of 8α,25-dihydroxy-3-oxoneocholecalciferol in liver

25-Hydroxycholecalciferol (25-OHD₃) is converted to 8α,25-dihydroxy-3-oxoneocholecalciferol [8,25-(OH)₂-3-oxone-D₃] by liver microsomes, alveolar macrophages and myeloid leukemia cells. The characteristics of this reaction in liver microsomes have been determined. Omission of an NADPH-generating system or NADH resulted in a >75% reduction in the production of 8,25-(OH)₂-3-oxone-D₃. In the absence of the cytosolic fraction, 25-OHD₃ was converted to products that comigrated with 8,25-(OH)₂-3-oxoneo-D₃ on a silica column developed with hexane-isopropanol, thereby preventing quantitation. Production of 8,25-(OH)₂-3-oxoneo-D₃ was unaffected by EDTA and was stimulated by N,N′-diphenyl-p-phenylenediamine. Both progesterone and pregnenolone inhibited production of 8,25-(OH)₂-3-oxoneo-D₃; inhibition by progesterone was greater than that by pregnenolone. 8,25-(OH)₂-3-Oxoneo-D₃ did not bind the thymus receptor for 1,25-dihydroxycholecalciferol [1,25-(OH)₂D₃] at concentrations 10-fold higher than that of 1,25-(OH)₂D₃. The lack of affinity of 8,25-(OH)₂-3-oxoneo-D₃ for the 1,25-(OH)₂D₃ receptor suggests that this metabolite is a degradative product of 25-OHD₃, which might be produced when 25-OHD₃ concentrations in the liver are excessive. Synthesis of this metabolite in the liver may be catalyzed by enzymes that also metabolize other steroids.     

25,26-dihydroxyvitamin D3 is not a major intermediate in 25-hydroxyvitamin D3-26,23-lactone formation

Structural similarities between 25S,26-dihydroxyvitamin D₃ and 25-hydroxyvitamin D₃-26,23-lactone and their concomitant multifold increase in the plasma of animals treated with pharmacological doses of vitamin D₃ suggest a precursor-product relationship. However, a single dose of 25S,26-[³H]dihydroxyvitamin D₃ given to rats treated chronically with pharmacological amounts of vitamin D₃ did not result in detectable plasma 25-[³H]hydroxyvitamin D₃-26,23-lactone. Multiple doses of synthetic 25S,26-dihydroxyvitamin D₃ given to vitamin D₃-deficient rats treated chronically with pharmacological amounts of vitamin D₂ also did not result in detectable plasma 25-hydroxyvitamin D₃-26,23-lactone. Furthermore, homogenates prepared from vitamin d-deficient chickens, dosed with 1,25-dihydroxyvitamin D₃, converted 25-[³H]hydroxyvitamin D₃ to 25-[³H]hydroxyvitamin D₃-26,23-lactone. But these same homogenates did not convert 25S,26-[³H]dihydroxyvitamin D₃ to 25-[³H]hydroxyvitamin D₃-26,23-lactone. These data indicate that 25,26-dihydroxyvitamin D₃ is not an intermediate in 25-hydroxyvitamin D₃26, 23-lactone formation.     

1,25-dihydroxyvitamin D3 specifically binds to a human breast cancer cell line (T47D) and stimulates growth

The T47D human breast cancer cell line contains a specific binding protein for 1.25-(OH)₂D₃, with 15000 sites per cell. The Kd (1.1 × 10^?10 M) and sedimentation coefficient on sucrose gradients (3.7S) are the same as those reported for the 1,25-(OH)₂D₃ receptor in other tissues. Other vitamin D₃ metabolites bound to the receptor with an order of affinities 1,25-(OH)₂D₃ > 1,24,25-(OH)₃D₃ > 25-OHD₃ > 24,25-(OH)₂D₃ > D₃. A new analogue 1β,25-(OH)₂D₃ was only as effective as 24,25-(OH)₂D₃ at displacing the hormone from the receptor. Cell growth was stimulated in a dose dependent manner by the addition of 1,25-(OH)₂D₃ (up to 0.8 nM) to the medium. A higher concentration of hormone was without effect.     

Biological activity assessment of the 26,23-lactones of 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 and their binding properties to chick intestinal receptor and plasma vitamin D binding protein

The binding of the natural and unnatural diastereoisomers 25-hydroxyvitamin D3-26,23-lactone and 1,25 dihydroxyvitamin D3-26,23-lactone to the vitamin D-binding protein (DBP) and 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] chick intestinal receptor have been investigated. Also, the biological activities, under in vivo conditions, of these compounds, in terms of intestinal calcium absorption (ICA) and bone calcium mobilization (BCM), in the chick are reported. The presence of the lactone ring in the C23-C26 position of the seco-steroid side chain increased two to three times the ability of both 25(OH)D3 and 1,25(OH)2D3 to displace 25(OH)[3H]D3 from the D-binding protein; however, the DBP could not distinguish between the various diastereoisomers. In contrast, the unnatural form (23R,25S) of the 25-hydroxy-lactone was found to be 10-fold more potent than the natural form, and the unnatural (23R,25S)1,25(OH)2D3-26,23-lactone three times more potent than the natural 1,25-dihydroxy-lactone in displacing 1,25(OH)2[3H]D3 from its intestinal receptor. While studying the biological activity of these lactone compounds, it was found that the natural form of the 25-hydroxy-lactone increased the intestinal calcium absorption 48 h after injection (16.25 nmol), while bone calcium mobilization was decreased by the same dose of the 25-hydroxy-lactone. The 1,25-dihydroxyvitamin D3-26,23-lactone in both its natural and unnatural forms was found to be active in stimulating ICA and BCM. These results suggest that the 25-hydroxy-lactone has some biological activity in the chick and that 1,25(OH)2D3-26,23-lactone can mediate ICA and BCM biological responses, probably through an interaction with 1,25-(OH)2D3 specific receptors in these target tissues.     

Competitive protein-binding radioassay of 24,25-dihydroxyvitamin D in sera from normal and anephric subjects

A competitive protein-binding radioassay for 24,25-dihydroxyvitamin D [24,25-(OH)₂D] in human serum has been developed. Whereas small amounts of [³H]24,25-(OH)₂D must be biosynthesized in order to trace the efficiency of the extraction and chromatographic procedures, tritiated 25-hydroxyvitamin D₃ ([³H]25-OHD₃) can be used as the assay tracer. Since 25-OHD₃ and 24,25-(OH)₂D₃ are equipotent in their competitive displacement of [³H]25-OHD₃ from rat serum, 25-OHD₃ can be used as the assay standard. Liquid-gel partition chromatography on small columns of Sephadex LH-20 can reliably isolate 24,25-(OH)₂D by batch elution. The purity of biosynthesized [³H]24,25-(OH)₂D₃ and the 24,25-(OH)₂D fraction isolated from serum was confirmed by high-pressure chromatography on 0.2 × 50 cm columns of 10-μm silica. Serum 24,25-(OH)₂D levels averaged 16% of the serum 25-OHD concentrations in normal subjects. Since chronic hemodialysis patients, without kidneys, had normal serum 24,25-(OH)₂D levels, significant extrarenal 25-hydroxycalciferol 24-hydroxylase activity occurs in these subjects. Since the present assay represents a reasonably simple extension of 25-OHD assay methodology, it should prove to be a useful technique in the analysis of clinical disorders of vitamin D metabolism.     

PTH(7-84) inhibits PTH(1-34)-induced 1,25-(OH)2D3 production in murine renal tubules

To elucidate whether PTH(7-84), a degradation product of PTH(1-84), which inhibits PTH(1-84)-induced bone resorption, also exerts an antagonistic effect on the kidney, we studied the effect of PTH(7-84) on PTH(1-34)-induced production of 1,25-(OH)₂D₃ in primary cultured murine renal tubules.Neonatal mouse renal tubules cultured in serum-free MEM for 7 days were treated with PTH(1-34) and/or PTH(7-84). Three hours after addition of 25-OHD₃ (10⁻⁶ M), 1,25-(OH)₂D₃ was determined. PTH(1-34) stimulated the conversion of 25-OHD₃ to 1,25-(OH)₂D₃, and PTH(7-84) dose-dependently inhibited this process. Real-time PCR revealed that PTH(1-34) increased the expression level of 1α-hydroxylase mRNA, whereas PTH(7-84) did not affect the expression level 1α or 24-hydroxylase mRNA.These in vitro data suggest that PTH(7-84) elicits an antagonistic effect in renal tubules through receptors different from the type I PTH/PTHrP receptor. This may at least partly account for the decreased serum level of 1,25-(OH)₂D in patients with severe primary hyperparathyroidism with renal failure.     

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.     

Ring A-stereoisomers of 1-hydroxyvitamin D3 and their relative binding affinities for the intestinal 1α,25-dihydroxyvitamin D3 receptor protein

A set of eight 1-hydroxyvitamin D₃ compounds comprising the four possible (5Z)-1,3-diol stereoisomers and the corresponding (5E)-double bond isomers, has been prepared in order to assess the effect of 1,3-diol stereochemistry and 5,6-double bond geometry on binding affinity for the intestinal 1,25-(OH)₂D₃-receptor protein. The compounds were synthesized from either vitamin D₃ or 3-epivitamin D₃ via 3,5-cyclovitamin D intermediates. Competitive receptor binding assays establish that all changes from the natural ring A-configuration (1S, 3R, 5Z) lead to decreased binding affinity, and confirm the importance of the 1-hydroxy function since the conversion of stereochemistry at that center from 1α(S) to 1β(R) has the most pronounced effect on binding affinity (attenuation by more than three orders of magnitude). Other modifications (i.e., conversion at C-3, or cis to trans isomerization of the 5,6-double bond) decrease binding affinity by more moderate (ca. 10-fold) but cumulative factors.     

Plasma 25-hydroxycholecalciferol and 1,25-dihydroxycholecalciferol concentrations are decreased in hind limb unloaded Dahl salt-sensitive female rats

Plasma 1,25-dihydroxyvitamin D (1,25-(OH)₂D) concentration was shown to decrease during bed rest in several studies when baseline plasma 25-hydroxyvitamin D (25-OHD) concentration was sub-optimal. Dahl salt-sensitive female (S) rats, but not Dahl salt-resistant female (R) rats, demonstrated a 50% decrease in plasma 1,25-dihydroxycholecalciferol (1,25-(OH)₂D₃) concentration after 28 days of hind limb unloading (HU, disuse model) during low salt intake (0.3%). We tested the vitamin D endocrine system response of female S rats to hind limb unloading during high salt intake (2%, twice that of standard rat chow to mimic salt intake in the USA). Hind limb unloading resulted in lower plasma 25-OHD₃ concentrations in S-HU rats than in R-HU rats (P < 0.05) and greater urinary loss of 25-OHD₃ by S-HU rats than by S rats (P < 0.05). Plasma 1,25-(OH)₂D₃ concentration of S-HU rats was half that of S rats, but was unchanged in R-HU rats. The association of low plasma 25-OHD concentration with decrease in plasma 1,25-(OH)₂D concentration of hind limb unloaded rats and of bed rest participants (published studies) suggests that low vitamin D status might be a risk factor for decrease in plasma vitamin D hormone concentration during long-term immobilization or bed rest.     

Isolation and identification of 23,25-dihydroxy-24-oxo-vitamin D3: A metabolite of vitamin D3

A new metabolite of vitamin D₃ has been isolated in pure form from incubations of rat kidney homogenates with 25-hydroxyvitamin D₃ [25-OH-D₃]. It was identified as 23,25-dihydroxy-24-oxo-vitamin D₃ [23,25(OH)₂-24-oxo-D₃] by means of ultraviolet absorption spectrophotometry and mass spectrometry. Also, 25-OH-D₃-26,23-lactone and 24R,25-dihydroxyvitamin D₃ were obtained from the same incubation mixtures. The enzyme activity responsible for the conversion of 25-OH-D₃ to 23,25(OH)₂-24-oxo-D₃ was induced by perfusion of the kidneys $\text{in}\text{vitro}$ with 50 nM 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃].     

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.     

Kidney mitochondrial metabolism of 25-hydroxyvitamin D3,: Evaluation of in vitro cation modulation

Oxidative phosphorylation and 1 α,25-dihydroxyvitamin D₃ [lα,25-(OH)₂D₃]synthesis in isolated mitochondria were decreased by the addition of strontium. Calcium effected a similar inhibition of 1α,25-(OH)₂D₃ synthesis which correlated with cation-induced mitochondrial swelling. The ultrastructural changes were found to be a consequence of experimental conditions and not a prerequisite for suppressed 1α,25-(OH)₂D₃ synthesis. Dietary administration of strontium or calcium also resulted in a decreased rate of 1α,25-(OH)₂D₃ synthesis; however, the decrease in 1-hydroxylase activity was accompanied by an induction of mitochondrial 25-hydroxyvitamin D₃ 24-hydroxylase activity. Such an in vivo-prompted mitochondrial response occurred in the absenee of morphological changes or extensive loss of oxidative phosphorylation activity. In contrast, no induction of 24-hydroxylase activity could be observed in acute studies using isolated mitochondria. Therefore, the in vitro action of calcium and strontium does not appear to reflect the in vivo mechanism whereby the cations act to change renal 25-hydroxyvitamin D₃ (25-OHD₃) hydroxylation. Results from in vitro studies corcerning the action of calcium to alter renal 25-OHD₃ metabolism should be interpreted in light of the cation's capacity to decrease oxidative phosphorylation and the subsequent intramitochondrial generation of NADPH.     

A specific binding protein for 1,25-dihydroxyvitamin D3 in rat intestinal cytosol.

In the presence of 0.3 M potassium chloride and 0.5 mM dithiothreitol, rat intestinal cytosol contains two binding proteins for 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃)¹ having sedimentation coefficients of 3.2S and 5–6S. The 3.2S protein is specific for 1,25-(OH)₂D₃ as determined by competition analysis, whereas the 5–6S protein binds 25-hydroxyvitamin D₃ (25-OH-D₃) exclusively.     

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.     

Comparative effects of some hydroxylated vitamin D metabolites on parathyrin secretion by dispersed rat parathyroid cells in vitro

We have previously discussed the action of 1 α,25-(OH)₂D₃, (24R) 24,25-(OH)₂ D₃ and (25S) 25,26-(OH)₂D₃ on parathyrin secretion by isolated rat parathyroid cells. In this work, we have compared these effects with those obtained with 1 α -OH D₃, 25-OH D₃ and 1 α -OH D₂.In decreasing order, the activities were : 1 α,25-(OH)₂D₃> 1 α -OH D₃ (24R) 24,25-(OH)₂D₃ > 25-OH D₃ > (25S) 25,26(OH)₂D₃> 1 α -OH D₂. The presence of two hydroxyl groups with one hydroxyl group in α position seems to have the higher activity to inhibit the parathyroid secretion. At least, the nature of the side chain conformation also plays a part upon the effect of PTH release.