Eberhard Mayer

Biological activity assessment of the vitamin D metabolites 1,25-dihydroxy-24-oxo-vitamin D3 and 1,23,25-trihydroxy-24-oxo-vitamin D3

Two new metabolites of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], namely 1,25(OH)2-24-oxo-vitamin D3 and 1,23,25(OH)3-24-oxo-vitamin D3, have been prepared in vitro using chick intestinal mucosal homogenates. To investigate the binding of 1,25(OH)2-[23-3H]-24-oxo-D3 and 1,23,25(OH)3-[23-3H]-24-oxo-D3 to the chick intestinal receptor we have isolated both metabolites in radioactive form using an incubation system containing 1,25(OH)2-[23,24-3H))-D3 with a specific radioactivity of 5.6 Ci/mmol. Both metabolites were highly purified by using Sephadex LH-20 chromatography followed by high-pressure liquid chromatography (HPLC). Sucrose density gradient sedimentation analysis showed specific binding of both tritium-labeled metabolites to the chick intestinal cytosol receptor. Experiments were carried out to determine the relative effectiveness of binding to the chick intestinal mucosa receptor for 1,25(OH)2D3. The results are expressed as relative competitive index (RCI), where the RCI is defined as 100 for 1,25(OH)2D3. Whereas the RCI obtained for 1,25(OH)2-24-oxo-D3 was 98 +/- 2 (SE), the RCI for 1,23,25(OH)3-24-oxo-D3 was only 28 +/- 6 (SE). Also, the biological activity of both new metabolites was assessed in vivo in the chick. In our assay for intestinal calcium absorption, 1,25(OH)2-24-oxo-D3 was active at a dose level of 1.63 and 4.88 nmol/bird (at 14 h), whereas 1,23,25(OH)3-24-oxo-D3 showed only weak biological activity in this system. In our assay for bone calcium mobilization, administration of both new metabolites showed modest activity at the 4.88-nmol dose level, which was reduced at the 1.63-nmol dose level. The results indicate that biological activity declines as 1,25(OH)2D3 is metabolized to 1,24R,25(OH)3D3, 1,25(OH)2-24-oxo-D3, and then 1,23,25(OH)3-24-oxo-D3.

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.

Study of 1,25-dihydroxyvitamin D3 induced alterations in the metabolism of [3H]25-hydroxyvitamin D3 using isolated perfused kidneys from D-sufficient rats.

Abstract 1α,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] induced alterations in the metabolism of [ 3 H]25-hydroxyvitamin D 3 [[ 3 H]25(OH)D 3 ] were studied using isolated perfused kidneys obtained from vitamin D-sufficient rats. Experimental perfused kidneys were exposed in vitro to 1,25(OH) 2 D 3 (5×10 −8 M) for 15 min and then washed with fresh perfusate before the metabolism of [ 3 H]25-(OH)D 3 was initiated. Control kidneys from the D-sufficient rats were studied without prior exposure to 1,25(OH) 2 D 3 . Tritiated 24R,25-dihydroxyvitamin D 3 [24,25(OH) 2 D 3 ] was the only metabolite produced by both the experimental and control kidneys during the initial two hours of perfusion. However, after two hours of perfusion, the experimental kidneys started producing one major and several minor new tritiated vitamin D metabolites; the production of these compounds then continued until the end of perfusion four hours later. Experimental kidneys, perfused with actinomycin D or cycloheximide failed to produce the new [ 3 H]-metabolites, but continued to produce only 24,25(OH) 2 D 3 similar to control kidneys. Thus the results suggest that 1,25(OH) 2 D 3 acts at the genome to induce the synthesis of enzymes needed for the production of new vitamin D metabolites in obtained kidneys from vitamin D replete rats.

Studies on the mode of action of calciferol—XXXV. Comparison of the biochemical properties and ligand specificities of receptors and antibodies for 1,25-dihydroxyvitamin D3☆☆☆

Abstract 1α,25(OH)2D3 is thought to act like a classical steroid hormone. It first associates with a cytoplasmic protein receptor in the target tissue. The cytoplasmic receptor—1α,25(OH)2D3-complex then moves to the nucleus where it alters transcriptional events and leads to a de novo synthesis of a vitamin D-dependent calcium binding protein. Receptors for 1α,25(OH)2D3 have been found in a wide number of mammalian and avian tissues, including the intestine, parathyroid glands, bone, kidney, pancreas, pituitary, epidermis and placenta. Vitamin D-dependent calcium-binding protein has been identified in a variety of tissues, e.g. intestine, kidney, pancreas, bone, adrenals, lungs, parathyroid glands and serum. Because of its high affinity for 1α,25(OH)2D3, the chick intestinal receptor is used in a steroid competition assay for determination of 1α,25(OH)2D3 from serum. The ligand specificity of this receptor and of anti-1α,25(OH)2D3-antisera which are employed in a radioimmunoassay for 1α,25(OH)2D3 are quantitatively compared in this paper.

Studies on the mode of action of calciferol: Comparison of the biochemical properties of an antiserum and the chick intestinal receptor both specific for 1,25-dihydroxyvitamin D3

Abstract The structural requirements for the interaction of 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] with an anti-1,25(OH) 2 D 3 antiserum and with the natural cytosolic receptor for 1,25(OH) 2 D 3 isolated from chick intestine have been evaluated quantitatively. The antiserum was raised in a rabbit against a 1,25(OH) 2 D 3 -hemisuccinate derivative which was linked to bovine serum albumin at the C-3 position of the steroid. For these cross-reaction studies structural analogs of 1,25(OH) 2 D 3 were used in competitive protein binding assays; their ability to interact with the binding proteins was expressed as relative competitive index (RCI) values where the RCI of 1,25(OH) 2 D 3 is defined to be 100. The results indicate that the 25-hydroxyl group is the most important hydroxyl for the interaction of 1,25(OH) 2 D 3 with this antiserum. The absence of this hydroxyl group decreases the RCI value to 0.2. Lack of the hydroxyl at carbon-3 or carbon-1 decreases the RCI value to 33 or 25, respectively, indicating that the specificity of this antiserum for the A ring is much lower than for the side chain. The high specificity for the side chain is underlined by the fact that insertion of an additional hydroxyl group at C-24 or C-26 of 1,25(OH) 2 D 3 decreases the binding affinity to the antiserum markedly. The chick intestinal mucosal receptor shows a comparable high specificity for the side chain of 1,25(OH) 2 D 3 , but an even higher specificity for the A ring in comparison to the antiserum. With the intestinal receptor, the 3-hydroxyl is only 1/ 10th as important as the 1-hydroxyl group and the 25-hydroxyl group for the binding process. Scatchard analysis showed a K D value of 1.7 × 10 −10 m for the antiserum and 2.3 × 10 −10 m for the chick intestinal mucosal receptor for the equilibrium binding of 1,25(OH) 2 D 3 at 2 °C. The association rate constant at 2 °C was determined to be 5.8 × 10 7 M −1 min −1 for the antiserum and 0.55 × 10 7 M −1 min −1 for the receptor, indicating a 10-fold more rapid association of 1,25(OH) 2 D 3 to the antiserum in comparison to the receptor. Furthermore, the dissociation process was found to be slower for the chick intestinal receptor (dissociation rate constant 3.6 × 10 −5 min −1 versus 21.0 × 10 −5 min −1 ).

24-Oxo and 26,23-lactone metabolites of 1,25-dihydroxyvitamin D3 have direct bone-resorbing activity

The biological activities of several 24-oxo and 26,23-lactone metabolites of vitamin D were determined in bone organ cultures. The 24-oxo metabolites were significantly more potent bone-resorbing agents than the lactones. 1,25-(OH)2-24-oxo-D3 had 0.18 X the bone-resorbing activity of 1,25-(OH)2D3 in fetal rat limb bones and was equipotent with 1,25-(OH)2D3 in neonatal mouse calvaria. In the limb bone system, 1,23,25-(OH)3-24-oxo-D3 had 0.08 X the activity of 1,25-(OH)2D3. 1,25-(OH)2D3 and 1,25-(OH)2-24-oxo-D3 had a similar time course of bone-resorbing effects in both bone culture systems. The most potent of the lactones, 1,25S-(OH)2D3-26,23R-lactone, had approximately 0.009 X the activity of 1,25-(OH)2D3 and approximately 500 times the activity of the 25S-OH-D3-26,23R-lactone. The 25S and 1,25S lactones were more potent than the 25R and 1,25R isomers. In experiments designed to determine whether either 1,25-(OH)2-24-oxo-D3 or 25R-OH-D3-26,23S-lactone could prevent the bone-resorbing activity of 1,25-(OH)2D3, no inhibitory effects were observed. The results suggest that conversion to the lactones represents a substantial inactivation step, whereas conversion to 24-oxo-derivatives results in less reduction in biological activity.

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

Abstract A new metabolite of vitamin D 3 has been isolated in pure form from incubations of rat kidney homogenates with 25-hydroxyvitamin D 3 [25-OH-D 3 ]. It was identified as 23,25-dihydroxy-24-oxo-vitamin D 3 [23,25(OH) 2 -24-oxo-D 3 ] by means of ultraviolet absorption spectrophotometry and mass spectrometry. Also, 25-OH-D 3 -26,23-lactone and 24R,25-dihydroxyvitamin D 3 were obtained from the same incubation mixtures. The enzyme activity responsible for the conversion of 25-OH-D 3 to 23,25(OH) 2 -24-oxo-D 3 was induced by perfusion of the kidneys in vitro with 50 nM 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ].

Mode of Action of 1α,25-Dihydroxyvitamin D

Vitamin D plays an essential role in calcium and phosphorus homeostasis. Before exerting biological activity, it must undergo metabolism (1–3); the major circulating form, 25-OH-D3, serves as a precursor for the production of 1,25(OH)2D3 or 24R,25(OH)2D3 in the kidney. Recent studies from our laboratory show that these two active forms of the vitamin can be further metabolized in the mammalian kidney. The metabolism of 24R,25(OH)2D3 leads through 25-OH-24-oxo-D3 and 23,25(OH)2-24- oxo-D3 to the cleavage of the side chain of the vitamin (4); l,25(OH)2D3 can be processed to yield either 1,24R,25(OH)3D3 or 1,25(OH)2D3-26,23- lactone (5). Whereas l,25(OH)2D3 and 24R,25(OH)2D3 are known as active forms of vitamin D (6), no biological role has yet been attributed to the other 21 metabolites that were chemically characterized (see Figure 1). l,25(OH)2D3 is the most active form known of vitamin D in terms of both calcium and phosphate absorption in the intestine and bone mineral mobilization. A current hypothesis concerning the mode of action of l,25(OH)2D3 in the target tissue is that the steroid associates first with a specific cytoplasmic receptor protein analogously to other steroid hormones. The cytoplasmic receptor-steroid complex then moves to the nucleus where it alters transcriptional events and leads to de novo synthesis of several proteins, of which vitamin D-dependent calcium binding protein is the most studied.