Chick Intestinal Cytosol Receptor Research Articles

Effect on carbon lengthening at the side chain terminal of 1.ALPHA.,25-dihydroxyvitamin D3 for calcium regulating activity.

A series of analogs of 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3 (1)] with alkyl substitutions in 26- and 27-positions have been tested for their activity 1) in competing with 1,25-(OH)2D3 for binding to chick intestinal cytosol receptor, 2) in ability for formation of multinucleated cells (MNC) with various osteoclastic cell characteristics from blast cells, and 3) in stimulating bone calcium mobilization in vitamin D-deficient rats. The relative potencies of 1,25-(OH)2D3, 1 alpha,25-dihydroxy-26,27-dimethylvitamin D3 (2), 1 alpha,25-dihydroxy-26,27-diethylvitamin D3 (3), and 1 alpha,25-dihydroxy-26,27-dipropylvitamin D3 (4) in competing for intestinal cytosolic binding were 1:1.1:0.25:0.05. The similar order of the abilities on formation of the multinucleated cells in the same series was observed. In a bone calcium mobilization test with vitamin D-deficient rats, 1 alpha,25-dihydroxy-26,27-dimethylvitamin D3 showed slightly less activity than 1,25-(OH)2D3 at 12 h after administration, but long lasting activity was observed during time course experiments. 1 alpha,25-Dihydroxy-26,27-diethylvitamin D3, and 1 alpha,25-dihydroxy-26,27-dipropylvitamin D3 were found to be much less active than 1,25-(OH)2D3 in a bone calcium mobilization test.

Identification and determination of 1 alpha,25-dihydroxyvitamin D3 in rat skin by high-performance liquid chromatography and radioreceptor assay.

Endogenous 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in normal rat skin was identified by using thermal isomerization to convert the metabolite into its pre-isomer, high-performance liquid chromatography (HPLC) and displacement potency with a chick intestinal cytosol receptor. When the metabolite in normal rat skin was determined by a radioreceptor assay after purification by Sep-Pak silica cartridge column chromatography and HPLC, the concentration was 71.0 +/- 6.6 pg/g of wet tissue (mean +/- S.D.). It is also shown that [3H]-1,25-(OH)2D3 and [3H]-25-hydroxyvitamin D3 administered intravenously in the mouse are located in the skin. These results suggest that the metabolite may play an important role in the skin.

The synthesis and biological activity of 25-hydroxy-26,27-dimethylvitamin D 3 and 1,25-dihydroxy-26,27-dimethylvitamin D 3: highly potent novel analogs of vitamin D 3

We synthesized 25-hydroxy-26,27-dimethylvitamin D 3, 9, and 1,25-dihydroxy-26,27-dimethylvitamin D 3,14, from chol-5-enic acid-3β-ol and tested their biological activity in vivo and in vitro. 9 was found to be a highly potent vitamin D analog with bioactivity similar to that of 25-hydroxyvitamin D 3. 9 bound to rat plasma vitamin D binding protein with approximately one-third the affinity of 25-hydroxyvitamin D 3. In a duodenal organ culture system and in a competitive binding assay with chick intestinal 1,25-dihydroxyvitamin D receptor, 9 was significantly more potent than 25-hydroxyvitamin D 3. 1,25-Dihydroxy-26,27-dimethylvitamin D 3, 14 was also highly active in vivo. At doses of 1000–5000 pmol/rat, its action was more sustained than that of 1,25-dihydroxyvitamin D 3.14 bound to vitamin 0 binding protein about 18 times less effectively than 1,25-dihydroxyvitamin D 3. 14 bound to the chick intestinal cytosol receptor with an affinity one-half that of 1,25-dihydroxyvitamin D 3. In a duodenal organ culture system, 14 was about half as active as 1,25-dihydroxyvitamin D 3. Extension of the sterol side chain, at C-26 and C-27, by methylene groups, prolongs the bioactivity of a vitamin D sterol hydroxylated at C-1 and C-25; the corresponding sterol, hydroxylated only at C-25, does not show any alteration of its bioactivity in vivo. These newly synthesized analogs may potentially be of therapeutic use in various mineral disorders.

1,25(OH)2D levels in dihydrotachysterol-treated patients: influence on 1,25(OH)2D assays.

Many clinicians continue to prefer dihydrotachysterol (DHT) as the initial vitamin D agent of choice in hypoparathyroidism and renal osteodystrophy because of its long history of efficacy and safety. Assessment of the factors influencing the clinical response to DHT treatment should include measurement of vitamin D metabolite profiles, but investigators have heretofore been unable to measure 1,25(OH)2D because levels have been found to be falsely elevated when employing the chick intestinal cytosol receptor assay. After converting from the chick cytosol receptor assay to the calf thymus receptor assay for measuring 1,25(OH)2D, we did not note falsely elevated levels of 1,25(OH)2D in DHT-treated patients. The design of this study, therefore, was aimed at determining whether or not the calf thymus receptor measured authentic 1,25(OH)2D in such patients. We controlled for the possibility that freezing and thawing or prolonged storage might have either lowered 1,25(OH)2D levels or degraded a metabolite(s) of DHT that would have otherwise been recognized as "1,25(OH)2D" by the calf receptor. Similarly, technical differences between the two assays, source of thymus, and potential interference by other cytosolic proteins were eliminated as causes for the difference between the 1,25(OH)2D levels in the two assays. Our experiments do not provide an explanation for why the thymus receptor does not "see" the interfering metabolite(s) of DHT. This could reflect either a tissue difference or perhaps a species difference. Our results do provide the first opportunity to expand the investigation of the metabolic effects of DHT therapy to include changes in intrinsic 1,25(OH)2D metabolism.

Synthesis, biologic activity, and protein binding characteristics of a new vitamin D analog, 22-hydroxyvitamin D 3

We synthesized a novel vitamin D analog, 22-hydroxyvitamin D 3 9 and tested its biologic activity (and antivitamin properties) in vivo in vitamin D-deficient rats, and in vitro in the chick embryonic duodenum. We examined its ability to bind to the sterol carrier protein, vitamin D binding protein and the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D 3. The new vitamin 9 was synthesized from 3β-hydroxy-22,23-dinorcholenic acid 1 in 12 steps. The vitamin 9 displayed no vitamin D agonist activity in the intestine or in bone in vivo and did not block the activity of vitamin D 3 or 25-hydroxyvitamin D 3. It was a weak vitamin D 3 agonist in the chick embryonal duodenum in vitro. It did not antagonize the activity of 1,25-dihydroxyvitamin D 3. Vitamin 9 bound to the chick intestinal cytosol receptor with low affinity. 22-Hydroxyvitamin D 3 and various vitamin D sterols were bound to vitamin D binding protein in the following order: 25-hydroxyvitamin D 3, (24R)-24,25-dihydroxyvitamin D 3, and (25S)-25,26-dihydroxyvitamin D 3 >22-hydroxyvitamin D 3 >11α -hydroxyvitamin D 3 >1,25-dihydroxy-vitamin D 3 >vitamin D 3. We conclude that the introduction of a hydroxyl group at C-22 in the side chain of the vitamin D 3 molecule decreases its biological activity.

Ectopic production of 1,25-dihydroxyvitamin D by B-cell lymphoma as a cause of hypercalcemia.

A patient with long-standing rheumatoid arthritis developed hypercalcemia (3.13 mmol/l) and was subsequently found to have a B-cell lymphoma (centroblastic type). The hypercalcemia was associated with high circulating concentrations of 1,25-dihydroxyvitamin D (235 pmol/l) and both abnormalities were corrected with treatment. A lymph node was excised before treatment and was incubated in vitro with either labeled or unlabeled 25-hydroxyvitamin D3. After purification of the extract and chromatography on three different HPLC systems, material comigrating with authentic 1,25-dihydroxyvitamin D3 was identified. This was shown to bind to a specific chick intestinal cytosol receptor and to dilute in parallel with synthetic 1,25-dihydroxyvitamin D3 in the receptor binding assay. In conclusion, hypercalcemia in malignant B-cell lymphoma can be due to extrarenal production of 1,25-dihydroxyvitamin D by lymphomatous tissue.

The difference of biological activity among four diastereoisomers of 1α,25-dihydroxycholecalciferol-26,23-lactone

All four possible diastereoisomers of 1α,25-dihydroxycholecalciferol-26,23-1αctone (1α,25-(OH) 2D 3-26,23-lactone) were chemically synthesized and were compared to 1α,25-dihydroxycholecalciferol (1α,25(OH) 2D 3) in terms of their stimu1ation, in vivo, of intestinal calcium transport and mobilization of calcium from bone in vitamin D-deficient rats (the two classic vitamin D-mediated responses), and their relative binding to the chick intestinal cytosol receptor for 1α,25-(OH) 2D 3. The receptor binding affinity results are expressed as relative competitive index (RCI), where the RCI is defined as 100 for 1α,25(OH) 2D 3. The RCI obtained for 23(S)25(S)-1α,25(OH) 2D 3-26,23-lactone was 7.90, for 23(R)25(R)-1α, 25(OH) 2D 3-26,23-lactone was 2.27, 23(S)25(R)-1α,25(OH) 2D 3-26,23-lactone was 0.17, for 23(R)25(S)-1α, 25(OH) 2D 3-26,23-lactone 0.22 and for the in vivo produced 1α,25(OH) 2D 3-26,23-lactone the RCI was only 0.17. Also the four diastereoisomers of 1α,25(OH) 2D 3-26,23-lactone all stimulated intestinal calcium transport, reaching a maximum 8 h after administration. Compared with the stimulation of intestinal calcium transport by 1α,25(OH) 2D 3, 23(S)25(S)-1α,25(OH) 2D 3-26,23-lactone was 1 4 as effective, 23(R)25(R)-1α,25(OH) 2D 3-26,23-1actone was 1 20 as effective, 23(S)25(R)-1α,25(OH) 2D 3-26,23-lactone was 1 74 as effective and 23(R)25(S)-1α,25(OH) 2D 3-26,23-lactone was 1 53 as effective. Similarly, 23(S)25(S)-1α,25(OH) 2D 3-26,23-lactone and 23(R)25(R)-1α,25(OH) 2D 3-26,23-lactone were estimated to be 3 and 20 times less active than 1α,25-(OH) 2D 3 in elevation of serum calcium. However, 23(S)25(R)-1α, 25(OH) 2D 3-26,23-1αctone and 23(R)25(S)-1α,25(OH) 2D 3-26,23-lactone decreased the serum calcium levels 24 h after administration. 23(S)25(R)-1α,25(OH) 2D 3-26,23-lactone reduced serum calcium concentrations to a greater extent than 23(R)25(S)-1α,25(OH) 2D 3-26,23-lactone. These results indicate that the biological activities of the diastereoisomers of 1α,25(OH) 2D 3-26,23-lactone were quite different among four stereochemical configurations.

The synthesis and biological activity of 22-fluorovitamin D 3: A new vitamin D analog

We synthesized 22-fluorovitamin D 3 from (22S) cholest-5-ene-3β, 22-diol-3β-acetate 2 . Compound 2 was treated with diethylaminosulfur trifluoride to give 22-fluorocholest-5-en-3β-acetate 3 and (E) 22-dehydrocholest-5-en-3β-acetate. Compound 3 was treated with N-bromosuccinimide to give a mixture of the respective 5,7- and 4,6-dienes. The 5,7-diene of 3 was separated from the 4,6-diene using the dienophile 4-phenyl-1,2,4-triazoline-3,5-dione. 22-Fluoro-5α, 8α-(3,5-dioxo-4-phenyl-1,2,4-triazolino)-cholest-6-en-3β-acetate 4 was purified by flash chromatography and treated with lithium aluminum hydride to generate 22-fluorocholesta-5,7-dien-3β-ol 5 . Photolysis of the diene 5 , followed by thermal equilibration, resulted in the synthesis of 22-fluorovitamin D 3 7 . The vitamin 7 increased active intestinal calcium transport only at a dose of 50,000 pmol/rat, whereas vitamin D 3 increased intestinal calcium transport at a dose of between 50 and 500 pmol/rat. 22-Fluorovitamin D 3 7 did not mobilize bone and soft tissue calcium at a dose as high as 50,000 pmol/rat, whereas vitamin D 3 was successful in doing so at a dose of 500 pmol/rat. When tested in the duodenal organ culture system, 22-fluorovitamin D 3 7 had approximately 1/25th the potency of vitamin D 3. It did not antagonize the activity of 1,25-dihydroxyvitamin D 3. 22-Fluorovitamin D 3 7 bound to the rat plasma vitamin D binding protein less avidly than vitamin D 3. 22-Fluorovitamin D 3 was bound very poorly to the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D 3. We conclude that the introduction of fluorine at the C-22 position results in a vitamin D sterol with decreased biologic activity when compared to vitamin D 3. The presence of a fluorine group at C-22 position inhibits the binding of the vitamin to rat vitamin D binding protein when compared to the binding of its hydrogen analog, vitamin D 3.

Synthesis of some side-chain homologues of 1 alpha,25-dihydroxyvitamin D3 and investigation of their biological activities.

In order to determine the structure of a new metabolite of 25-hydroxy-24-epivitamin D2, 1, 25-dihydroxy-22-dehydro-24-homo- and 26-homo-vitamin D3 were synthesized. The saturated analogues at the C-22 position of these homovitamin D3 compounds were also synthesized. For the construction of the side chain of these homo-analogues, the orthoester Claisen rearrangement of the allylic alcohol was employed.Assay of binding of these compounds with the chick intestinal cytosol receptor protein for 1, 25-(OH)2D3 was carried out by the displacement method. The results indicated that these homovitamin D3 analogues are as active as 1, 25-(OH)2D3. The bone calcium-mobilizing activities of these compounds were equivalent to that of 1, 25-(OH)2D3.

Synthesis and biological activity of 3β-fluorovitamin D 3: Comparison of the biological activity of 3β -fluorovitamin D 3 and 3-deoxyvitamin D 3

The alteration in the biologic activity of the vitamin D 3 molecule resulting from the replacement of a hydrogen atom with a fluorine atom is a subject of fundamental interest. To investigate this problem we synthesized 3β -fluorovitamin D 3 6 and its hydrogen analog, 3-deoxyvitarnin D 3 7, and tested the biologic activity of each by in vitro and in vivo methods. Contrary to previous reports which showed that 3β-fluorovitamin D 3 was as active as vitamin D 3 in vivo, we found that the fluoro-analog was less active than vitamin D 3. With regard to stimulation of intestinal calcium transport and bone calcium mobilization in the D-deficient hypocalcemic rat, 3β-fluorovitamin D 3 snowed significantly greater biologic activity than its hydrogen analog, 3-deoxyvitamin D 3. In the organ-cultured, embryonic chick duodenum, 3β-fluorovitamin D 3 was approx 1 1000 th as active as the native hormone, 1,25-dihydroxyvitamin D 3, while 3-deoxyvitamin D 3 was inactive even at μ M concentrations, in the induction of the vitamin D-dependent, calcium-binding protein. With regard to in vitro activity in displacing radiolabeled 25-hydroxyvitamin D 3 from vitamin D binding protein and radiolabelled 1,25-dihydroxyvitamin D 3 from a chick intestinal cytosol receptor, 3β-fluorovitamin D 3 and 3β-deoxyvitamin D 3 both showed very poor binding efficiencies when compared with vitamin D 3. Our results show that the substitution of a fluorine atom for a hydrogen atom at the C-3 position of the vitamin D 3 molecule results in a fluorovitamin 6 with significantly more biological activity than its hydrogen analog, 3-deoxyvitamin D 3 7.

26,26,26,27,27,27-Hexafluoro-1,25-dihydroxyvitamin D 3: A highly potent, long-lasting analog of 1,25-dihydroxyvitamin D 3

A new fluoro analog of 1,25-dihydroxyvitamin D 3, i.e., 26,26,26,27,27,27-hexafluoro-1, 25-dihydroxyvitamin D 3, has been compared with the native hormone, 1,25-dihydroxyvitamin D 3, in its biological potency, duration of action, and binding to the vitamin D transport protein and intestinal receptor protein. The fluoro analog is about 5 times more active than the native hormone in healing rickets and elevating serum inorganic phosphorus levels of rachitic rats. It is about 10 times more active than 1,25-dihydroxyvitamin D 3 in increasing intestinal calcium transport and bone calcium mobilization of vitamin D-deficient rats fed a low-calcium diet. Furthermore, the higher biopotency is manifested in animals after oral dosing. Of great importance is that the action of the fluoro analog is longer lasting than that of 1,25-dihydroxyvitamin D 3. This is especially apparent in the elevation of serum phosphorus and bone mineralization responses. The fluoro analog is only slightly less competent than 1,25-dihydroxyvitamin D 3 in binding to the vitamin D transport protein in rat blood, and is one-third as competent as 1,25-dihydroxyvitamin D 3 in binding to the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D 3. These results suggest that the basis for increased potency of this analog is likely the result of less rapid metabolism.

Biological activity assessment of 1α,25-dihydroxyvitamin D 3-26,23-lactone in the rat

1α,25-Dihydroxyvitamin D 3-26,23-lactone [1α,25(OH) 2D 3-26, 23-lactone] was compared to 1α,25-dihydroxyvitamin D 3 [1α,25(OH) 2D 3] in terms of their stimulation, in vivo, of intestinal calcium transport and mobilization of calcium from bone in the rat (the two classic vitamin D-mediated responses), and their relative binding to the chick intestinal receptor for 1α,25(OH) 2D 3. 1α,25-(OH) 2D 3-26,23-lactone was found to be only one-thirtieth as active as 1α,25-(OH) 2D 3 in the stimulation of intestinal calcium transport and was found to mediate a significant reduction in the steady-state serum calcium levels. Associated with the reduction in serum calcium was a significant increase in urinary calcium excretion for 24 h after the administration of the steroid. Prior administration of 1α,25(OH) 2D 3-26,23-lactone partially blocked the actions of a subsequently administered dose of 1α,25(OH) 2D 3 in increasing serum calcium levels, but did not affect the action of 1α,25(OH) 2D 3 in stimulating intestinal calcium transport. The binding affinity of 1α,25(OH) 2D 3-26,23-lactone to the chick intestinal cytosol receptor protein was observed to be 670 times lower than that of 1,25-(OH) 2D 3 which indicates that perturbation of the 25-hydroxylated side chain by formation of the 26,23-lactone causes a significant reduction in ligand affinity for the receptor.

Synthesis and biological activity of (22E,24R)- and (22E,24S)-1 alpha,24-dihydroxy-22-dehydrovitamin D3.

Chemical synthesis of (22E, 24R)- and (22E, 24S)-1, 24-dihydroxy-Δ22-vitamin D3 has been achieved starting with the commercially available dinorcholenic acid acetate. Synthesis involved introduction of the 1-hydroxy group by a reduction of the 1, 2-epoxide generated by epoxidation of the 1, 4, 6-trien-3-one. The side chain on the steroid was then constructed by means of a Wittig reaction followed by introduction of the Δ7 bond by standard methods and its protection with 1-phenyl-1, 2, 4-triazoline-3, 5-dione. Subsequent reduction of the hydroxy groups in the steroid side chain followed by reduction of the Diels-Alder addition products yielded the both 24-isomers. The 5, 7-dienes were irradiated and the corresponding vitamin D compounds isolated. Nuclear magnetic resonance was used to identify individual isomers. The (22E, 24S)-1, 24-hydroxyvitamin D3 compound bound equally well to the chick intestinal cytosol receptor as 1, 25-dihydroxyvitamin D3, while the 24R-isomer was approximately ten times less active. In vivo, both isomers were less active than 1, 25-dihydroxyvitamin D3 ; however, the 24S-isomer was considerably more active than the 24R-isomer approaching the activity of 1, 25-dihydroxyvitamin D3.

Metabolism and binding properties of 24,24-difluoro-25-hydroxyvitamin D 3

To evaluate possible functional roles for 24,25-dihydroxyvitamin D 3, 24,24-difluoro-25-hydroxyvitamin D 3 has been synthesized and shown to be equally as active as 25-hydroxyvitamin D 3 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 D 3 was detected in the serum obtained from vitamin D-deficient rats that had been given 24,24-difluoro-25-hydroxyvitamin D 3, while large amounts were found when 25-hydroxyvitamin D 3 was given. Incubation of the 24,24-difluoro compound with kidney homogenate prepared from vitamin D-replete chickens failed to produce 24,25-dihydroxyvitamin D 3, while the same preparations produced large amounts of 24,25-dihydroxyvitamin D 3 from 25-hydroxyvitamin D 3. Kidney homogenate prepared from vitamin D-deficient chickens produced 24,24-difluoro-1,25-dihydroxyvitamin D 3 from 24,24-difluoro-25-hydroxyvitamin D 3 and 1,25-dihydroxyvitamin D 3 from 25-hydroxyvitamin D 3. In binding to the plasma transport protein for vitamin D compounds, 24,24-difluoro-25-hydroxyvitamin D 3 is less active than 25-hydroxyvitamin D 3 and 24 R,25-dihydroxyvitamin D 3. In binding to the chick intestinal cytosol receptor, 24,24-difluoro-25-hydroxyvitamin D 3 is more active than 25-hydroxyvitamin D 3 which is itself more active than 24 R,25-dihydroxyvitamin D 3. The 24,24-difluoro-1,25-dihydroxyvitamin D 3 is equal to 1,25-dihydroxyvitamin D 3, and both are 10 times more active than 1,24 R,25-trihydroxyvitamin D 3 in this system. These results provide strong evidence that the C-24 carbon of 24,24-difluoro-25-hydroxyvitamin D 3 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.

Biological activity assessment of the vitamin D metabolites 1,25-dihydroxy-24-oxo-vitamin D 3 and 1,23,25-trihydroxy-24-oxo-vitamin D 3

Two new metabolites of 1,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3], namely 1,25(OH) 2-24-oxo-vitamin D 3 and 1,23,25(OH) 3-24-oxo-vitamin D 3, have been prepared in vitro using chick intestinal mucosal homogenates. To investigate the binding of 1,25(OH) 2-[23- 3H]-24-oxo-D 3 and 1,23,25(OH) 3-[23- 3H]-24-oxo-D 3 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]-D 3 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) 2D 3. The results are expressed as relative competitive index (RCI), where the RCI is defined as 100 for 1,25(OH) 2D 3. Whereas the RCI obtained for 1,25(OH) 2-24-oxo-D 3 was 98 ± 2 (SE), the RCI for 1,23,25(OH) 3-24-oxo-D 3 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-D 3 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-D 3 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) 2D 3 is metabolized to 1,24 R,25(OH) 3D 3) 1,25(OH) 2-24-oxo-D 3, and then 1,23,25(OH) 3-24-oxo-D 3.

An in vitro study of the stability of the chicken intestinal cytosol 1,25-dihydroxyvitamin D 3-specific receptor

The stability of the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D 3 has been examined using radiological binding studies and sucrose density gradient ultracentrifugation. Specific binding of 1,25-dihydroxyvitamin D 3 to the 3.7 S binding protein decreases in crude cytosol in a time- and temperature-dependent manner. Increased receptor instability is also observed outside a pH range of 6 to 10. Ionic strength does not seem to be a critical factor in preventing loss of specific 1,25-dihydroxyvitamin D 3 binding activity. However, when KCl is present at a concentration of 300 m m during cytosol preparation, quantitatively more specific binding per unit protein was obtained. Consistent with the idea that loss of specific binding might be due to enzymatic degradation or inactivation of receptor, dilution of cytosol was found to slow the rate of loss of specific 1,25-dihydroxyvitamin D 3 binding. The importance of maintaining a reducing environment for the 1,25-dihydroxyvitamin D 3 binding protein is demonstrated by the destruction of binding activity by n-ethylmaleimide and by the increased stability in the presence of 5.0 m m dithiothreitol. Likewise, 5.0 m m monothioglycerol was partially effective in preventing the loss of specific 1,25-dihydroxyvitamin D 3 binding during in vitro incubation. Several protease inhibitors were not able to exert a stabilizing influence on receptor integrity during in vitro incubations. Albeit, both tosylamide-phenylethylchloromethyl ketone and p-hydroxymercuribenzoate actually decreased specific 1,25-dihydroxyvitamin D 3 binding. This inhibition appeared to be reversible if samples were subsequently incubated in the presence of dithiothreitol. These results clearly demonstrate that the aporeceptor is extremely unstable and the integrity of sulfhydryl constituents is of primary importance.

Evidence for the metabolism of 24 R-hydroxy-25-fluorovitamin D 3 and 1α-hydroxy-25-fluorovitamin D 3 to 1α,24 R-dihydroxy-25-fluorovitamin D 3

High-pressure liquid chromatography capable of resolving all known vitamin D metabolites and a sensitive competitive binding protein assay specific for 1α,25-dihydroxyvitamin D 3 were used to assay the blood of rats dosed with ethanol, 1α-hydroxyvitamin D 3, 24 R-hydroxy-25-fluorovitamin D 3, or 1α-hydroxy-25-fluorovitamin D 3. Compared to the ethanoldosed animals, the blood of rats dosed with 1α-hydroxyvitamin D 3 had increased levels of 1α,25-dihydroxyvitamin D 3; but those dosed with the fluorinated vitamins did not. Instead, their blood contained a compound that cochromatographs with 1α,24 R-dihydroxyvitamin D 3 on high-pressure liquid chromatography and binds to the 1,25-dihydroxyvitamin D 3 receptor proteins. 1α,24 R-Dihydroxyvitamin D 3 binds as well as 1α, 25-dihydroxyvitamin D 3 to the chick-intestinal cytosol receptor protein for 1α,25-dihydroxyvitamin D 3; whereas 1α,24 S-dihydroxyvitamin D 3 binds only one-tenth as well as 1α,25-dihydroxyvitamin D 3. Thus it appears that in vivo, the fluorinated vitamin D compounds are converted to a compound likely to be 1α,24 R-dihydroxy-25-fluorovitamin D 3 and that may rival the potency of 1α,25-dihydroxyvitamin D 3.

Intestinal 1,25-dihydroxyvitamin D 3 binding protein: Specificity of binding

The binding of metabolites of vitamin D and their analogs to the 3.7S chick intestinal cytosol receptor protein has been specifically studied by competitive binding techniques and polyethylene glycol precipitation of the complex. The structural requirements for the interaction between the vitamin D molecule and the receptor could be assessed without the nuclear chromatin binding step. These measurements have shown that 1,25-dihydroxyvitamin D 3 and 1,25-dihydroxyvitamin D 2 are equally competitive and are the most active. Of the structural features of the compounds, the 1α-hydroxyl is most important followed by the 25-hydroxyl and the 3β-hydroxyl. The addition of a second hydroxyl near carbon 25 markedly reduces binding whether on the 26 carbon or the 24 carbon. A hydroxyl on C-24 could substitute to some degree for the 25-hydroxyl inasmuch as 24-hydroxyvitamin D 3 was much more effective than vitamin D 3 but less effective than 25-hydroxyvitamin D 3. In general the patterns of binding affinities correlated well with the biological activity of the various analogs strongly supporting a physiological role for the 1,25-dihydroxyvitamin D 3 binding protein. It also suggests that of the two-step receptor mechanism, the structural specificity is located in the initial interaction of the 1,25-dihydroxyvitamin D 3 and the cytosol receptor.