Studies on the role of 1,25-dihydroxyvitamin D in chick embryonic development

Vitamin D-deficient laying hens were repleted with 25-hydroxy[26,27-3H]vitamin D3 or 1,25-dihydroxy[26,27-3H]vitamin D3. Egg production returned to normal for both groups of hens by the third week. Eggs from hens fed either 25-hydroxy[26,27-3H]vitamin D3 or 1,25-dihydroxy[26,27-3H]vitamin D3 contained 1,25-dihydroxy[26,27-3H]vitamin D3. Eggs from hens fed 25-hydroxy[26,27-3H]vitamin D3 contained substantial amounts of 25-hydroxy[26,27-3H]vitamin D3, while those from hens fed 1,25-dihydroxy[26,27-3H]vitamin D3 contained none. Plasma from 18-day embryos from hens fed 1,25-dihydroxy[26,27-3H]vitamin D3 contained little or no 1,25-dihydroxy[26,27-3H]vitamin D3, while that from 18-day embryos from hens given 25-hydroxy[26,27-3H]vitamin D3 had normal levels of 1,25-dihydroxy[26,27-3H]vitamin D3. No eggs from hens fed 1,25-dihydroxy[26,27-3H]vitamin D3 hatched, while eggs from hens fed 25-hydroxy[26,27-3H]vitamin D3 achieved a hatchability of 90%. It appears that embryos from hens maintained on 1,25-dihydroxyvitamin D3 as their sole source of vitamin D are essentially vitamin D deficient.     

Effect of vitamin D3 derivatives on cholesterol synthesis and HMG-CoA reductase activity in cultured cells

Treatment of logarithmically growing rat intestinal epithelial cells (IEC-6) in culture with vitamin D3 (cholecalciferol), 25-hydroxy vitamin D3 (25-hydroxy cholecalciferol), 1,25-dihydroxy vitamin D3 (1,25-dihydroxycholecalciferol), and 24,25 dihydroxy vitamin D3 (24(R),25-dihydroxycholecalciferol), caused an inhibition of the cholesterol biosynthetic pathway at two separate sites. At concentrations greater than 2 micrograms/ml, the hydroxylated forms of vitamin D3 caused an accumulation of methyl sterols indicating an inhibition of lanosterol demethylation. Vitamin D3, however, had little effect on lanosterol demethylation. A second site of inhibition occurs at 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), the rate limiting enzyme in cholesterol biosynthesis at concentrations less than 2 micrograms/ml. All vitamin D3 compounds, except 1,25-dihydroxy vitamin D3, inhibited HMG-CoA reductase activity in a concentration-dependent manner. The lack of inhibition of HMG-CoA reductase activity by 1,25-dihydroxy vitamin D3 in IEC-6 cells was not due to impaired uptake, since 1,25-dihydroxy vitamin D3 caused an accumulation of methyl sterols under similar conditions. The inhibition of HMG-CoA reductase activity and cholesterol synthesis by vitamin D3 and 25-hydroxy vitamin D3 was also observed in other cell culture lines such as human skin fibroblasts (GM-43), transformed human liver cells (Hep G2), and mouse peritoneal macrophages (J-774). On the other hand, 1,25-hydroxy vitamin D3 showed effects on HMG-CoA reductase activity that varied with the cell line. In J-774 and human skin fibroblasts, 1,25-dihydroxy vitamin D3 showed a biphasic effect on reductase activity such that at low concentrations reductase activity was inhibited but was restored to control values at high concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of 1,25-dihydroxyvitamin D3: evidence for side-chain oxidation.

Approximately 7% of a 650-pmol dose of 25-hydroxyl[26,27-14C]vitamin D3 and 25% of a 325-pmol dose of 1,25-dihydroxyl[26,27-14C]vitamin D3 are metabolized to 14CO2 by vitamin D deficient rats. Nephrectomy prevents the metabolism of 25-hydroxy[26,27-14C]vitamin D3 to 14CO2 but not that of 1,25-dihydroxy[26,27-14C]vitamin D3. Less than 5% of the 14C from 24,25-dihydroxy[26,27-14C]vitamin D3 is metabolized to 14CO2. Feeding diets high in calcium and supplemented with vitamin D3 markedly diminishes the amount of 14CO2 formed from 25-hydroxy[26,27-14C]vitamin D3 but not that from 1,25-dihydroxyl[26,27-14C]vitamin D3. These results provide strong evidence that only 1-hydroxylated vitamin D compounds and especially 1,25-dihydroxyvitamin D3 undergo side-chain oxidation and cleavage to yield an unknown metabolite and CO2.     

A rapid assay for 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D 24-hydroxylase

A rapid method for the measurement of the 24-hydroxylated metabolites of 25-hydroxy[26,27-3H]vitamin D3 and 1,25-dihydroxy[26,27-3H]vitamin D3 has been developed. This measurement has, in turn, made possible a rapid assay for the 24-hydroxylases of the vitamin D system. The assay involves the use of 26,27-3H-labeled 1,25-dihydroxyvitamin D3 or 25-hydroxyvitamin D3 as the substrate and treatment of the enzyme reaction mixture with sodium periodate, which specifically cleaves the 24-hydroxylated products between carbons 24 and 25, releasing tritiated acetone. The acetone is measured after its separation from the labeled substrate by using a reversed-phase cartridge. The results obtained with this assay were validated by comparison with the results obtained with a well-established high-performance liquid chromatography assay. The activity of the enzyme determined by both methods was equal. This assay has been successfully used for the rapid screening of column fractions during purification of the enzyme and in the screening for monoclonal antibodies to the 24-hydroxylase.     

1 alpha-hydroxy-25-fluorovitamin D3: a potent analogue of 1 alpha,25-dihydroxyvitamin D3

Chemically synthesized 1 alpha-hydroxy-25-fluorovitamin D3 was compared to 1,25-dihydroxyvitamin D3 for potency in the chick intestinal cytosol-binding protein assay, induction of intestinal calcium transport, mobilization of calcium from bone, and epiphyseal plate calcification in the rat. The 25-fluorinated analogue causes 50% displacement of 1,25-dihydroxy[23,24-3H]D3 at 1.8 X 10(-8) M in the competitive protein-binding assay, whereas only 5.6 X 10(-11) M of unlabeled 1,25-dihydroxyvitamin D3 is needed for equal competition. This 315-fold difference between and 1 alpha-hydroxy-25-fluorovitamin D3 indicates that the fluoro analogue is about equipotent with 1 alpha-hydroxyvitamin D3 in the protein-binding assay. However, 1 alpha-hydroxy-25-fluorovitamin D3 is 1/50 as active as 1,25-dihydroxyvitamin D3 in vivo in the stimulation of intestinal calcium transport and bone calcium mobilization in vitamin D deficient rats on a low-calcium diet. Likewise, 1 alpha-hydroxy-25-fluorovitamin D3 is about 40 times less active than 1,25-dihydroxyvitamin D3 in inducing endochondrial calcification in rachitic rats. No selective actions of 1alpha-hydroxy-25-fluorovitamin D3 were noted. Since the 25 position of the analogue is blocked by a fluorine atom, it appears that 25-hydroxylation of 1 alpha-hydroxylated vitamin D compounds in vivo is not an obligatory requirement for appreciable vitamin D activity.     

Specific binding protein for 1,25-dihydroxyvitamin D3 in bovine mammary gland

Specific binding proteins for 1,25-dihydroxyvitamin D₃ were identified in bovine mammary tissue obtained from lactating and non-lactating mammary glands by sucrose density gradient centrifugation. The macromolecules had characteristic sedimentation coefficients of 3.5-3.7 S. The interaction of l,25-dihydroxy[³H]vitamin D₃ with the macromolecule of the mammary gland cytosol occurred at low concentrations, was saturable, and was a high affinity interaction (K_d = 4.2 × 10^?10M at 25 °C). Binding was reversed by excess unlabeled 1,25-dihydroxyvitamin D₃, was destroyed by heat and/or incubation with trypsin. It is thus inferred that this macromolecule is protein as it is not destroyed by ribonuclease or deoxyribonuclease. 25-hydroxyvitamin D₃, 24,25-dihydroxyvitamin D₃, and vitamin D₃ did not effectively compete with 1,25-dihydroxyvitamin D₃ for binding to cytosol of mammary tissue at near physiological concentrations of these analogs, thus demonstrating the specificity of the binding protein for 1,25-dihydroxyvitamin D₃. In vitro subcellular distribution of 1,25-dihydroxy[³H]vitamin D₃ demonstrated a time- and temperature-dependent movement of the hormone from the cytoplasm to the nucleus. By 90 min at 25 °C 72% of the 1,25-dihydroxy[³H]vitamin D₃ was associated with the nucleus. In addition a 5–6 S macromolecule which binds 25-hydroxy[³H]vitamin D₃ was demonstrated in mammary tissue. Finally, it is possible that the receptor-hormone complex present in mammary tissue may function in a manner analogous to intestinal tissue, resulting in the control of calcium transport by 1,25-dihydroxyvitamin D₃ in this tissue.     

Isolation and characterization of 1 alpha-hydroxy-23-carboxytetranorvitamin D: a major metabolite of 1,25-dihydroxyvitamin D3.

The in vivo side-chain oxidation of 1 alpha,25-dihydroxyvitamin D3 was investigated by using a double-label radiotracer technique. Rats dosed with 1 alpha,25-dihydroxy-[3 alpha-3H]vitamin D3 and 1 alpha,25-dihydroxy[26,27-14C]vitamin D3 produced compounds with a high 3H/14C ratio. These compounds were found in sizable quantities in intestine and liver within 3 h after dosing. The major side-chain oxidized metabolite migrated as an acid on DEAE-Sephadex chromatography and contained no 14C. Methyl esterification of this compound with diazomethane proceeded in good yield and rendered the compound more amenable to chromatographic purification. The metabolite was isolated in several steps from rats dosed with 1 microgram of 1 alpha,25-dihydroxy[3 alpha-3H]vitamin D3. The metabolite was obtained in pure form as the methyl ester and was positively identified as 1 alpha,3 beta-dihydroxy-24-nor-9,10-seco-5,7,10(19)cholatrien-23-oic acid. The trivial name calcitroic acid is proposed for this major side-chain oxidized metabolite of 1,25-dihydroxyvitamin D3.     

A binding assay for 25-hydroxycalciferols and 24R,25-dihydroxycalciferols using bovine plasma globulin

The concentrations of the 25-hydroxy and 24R,25-dihydroxy derivatives of vitamin D were determined in 100 μ1 plasma samples using calciferol binding globulin from bovine plasma. Sufficient quantities of 24R,25-dihydroxy vitamin D were found in bovine, porcine, chicken and human plasma to interfere in the assay of 25-hydroxy vitamin D in unfractionated extracts. No metabolites of vitamin D could be found in rainbow trout plasma.     

Studies on the site of 1,25-dihydroxyvitamin D3 synthesis in vivo

Anephric, vitamin D-deficient male rats were injected with a physiologic dose of 25-hydroxy[26,27-3H]vitamin D3 (specific activity of 160 Ci/mmol), and 18-20 h later, intestine, bone, and serum were analyzed by high performance liquid chromatography for 1,25-dihydroxy-[26,27-3H]vitamin D3. Identical studies were carried out using sham-operated rats and rats with ligated ureters. No 1,25-dihydroxy[26,27-3H]vitamin D3 was detected in the tissues from anephric rats, while large amounts were detected in sham-operated and ureteric ligated controls. This result demonstrates that in the nonpregnant rat, 1,25-dihydroxyvitamin D3 is either not synthesized or is synthesized in vanishingly small amounts in bone and intestine in vivo, casting considerable doubt of the physiological importance of reports of in vitro synthesis of 1,25-dihydroxyvitamin D3 by cells in culture derived from bone and elsewhere.     

Metabolism and biologica.

24R,24,25-Dihydroxyvitamin D3 is capable of inducing a minimal intestinal calcium transport response in chicks when compared to an equal amount of 25-hydroxyvitamin D3. 1,24,25-Trihydroxyvitamin D3 is also less active than 1,25-dihydroxyvitamin D3, and its activity is much shorter lived than that of 1,25-dihydroxyvitamin D3. A comparison of the metabolism of 25-hydroxy[26,27-3H]vitamin D3 and 24,25-dihydroxy[26,27-3H]vitamin D3 in the rat and chick shows that 24,25-dihydroxyvitamin D3 and 1,24,25-trihydroxyvitamin D3 disappear at least 10 times more rapidly from the blood and intestine of chicks. Furthermore, examination of the excretory products from both of these species demonstrates that chicks receiving a single dose of 24,25-dihydroxy[26,27-3H]vitamin D3 excrete 66% of the total radioactivity by 48 hours, whereas rats receiving the same dose excrete less than one-half that amount. These results demonstrate that 24,25-dihydroxyvitamin D3 is considerably less biologically active in the chick than in the rat, probably due to more rapid metabolism and excretion.     

Vitamin D and chick embryonic yolk calcium mobilization: identification and regulation of expression of vitamin D-dependent Ca2(+)-binding protein, calbindin-D28K, in the yolk sac

The developing chick embryo acquires calcium from two sources. Until about Day 10 of incubation, the yolk is the only source; thereafter, calcium is also mobilized from the eggshell. We have previously shown that during normal chick embryonic development, vitamin D is involved in regulating yolk calcium mobilization, whereas vitamin K is required for eggshell calcium translocation by the chorioallantoic membrane. We have studied here the biochemical action of 1,25-dihydroxy vitamin D3 in the yolk sac by examining the expression and regulation of the cytosolic vitamin D-dependent calcium-binding protein, calbindin-D28K. Two types of embryos are used for this study, normal embryos developing in ovo and embryos maintained in long-term shell-less culture ex ovo, the latter being dependent solely on the yolk as their calcium source. Our findings are (1) calbindin-D28K is expressed in the embryonic yolk sac, detectable at incubation Days 9 and 14; (2) the embryonic yolk sac calbindin-D28K resembles that of the adult duodenum in both molecular weight (Mr 28,000) and isoelectric point, as well as the presence of E-F hand Ca2(+)-binding structural domains; (3) systemic calcium deficiency caused by shell-less culture of chick embryos results in enhanced expression of calbindin-D28K in the yolk sac during late development; (4) yolk sac calbindin-D28K expression is inducible by 1,25-dihydroxy vitamin D3 treatment in vivo and in vitro; and (5) immunohistochemistry revealed that yolk sac calbindin-D28K is localized exclusively to the cytoplasm of the yolk sac endoderm. These findings indicate that the chick embryonic yolk sac is a genuine target tissue of 1,25-dihydroxy vitamin D3.     

25-Hydroxylation of 1 alpha-hydroxyvitamin D-3 in rat and human liver

1 alpha-Hydroxyvitamin D-3 25-hydroxylase activity was measured in subcellular fractions of rat and human liver. The formation of 1,25-dihydroxyvitamin D-3 was determined by high pressure liquid chromatography. In rat liver 1 alpha-hydroxyvitamin D-3 25-hydroxylase activities were found in the purified nuclei, the heavy mitochondrial fraction and the microsomal fraction. The enrichment of 25-hydroxylase activity was highest in the heavy mitochondrial fraction. With this fraction a minimum amount (about 0.5 mg) of protein was required before formation of 1,25-dihydroxyvitamin D-3 could be detected. Above this amount the reaction was linear with amount of protein up to at least 2 mg/ml. The reaction was also linear with time up to 60 min. An apparent Km value of 2 X 10(-5) M was found. The mitochondrial 25-hydroxylase was stimulated by addition of cytosolic protein or bovine serum albumin. The degree of stimulation was dependent on the amount of mitochondrial protein present in the incubation mixture. Maximal stimulation was seen with 0.2 mg/ml of either protein in the presence of 0.5 mg mitochondrial protein. The stimulating effect remained after heating the protein for 5 min at 100 degrees C. The cytosolic protein did not stimulate a reconstituted mitochondrial 1 alpha-hydroxyvitamin D-3 25-hydroxylase. The mitochondrial vitamin D-3 25-hydroxylase was inhibited both by cytosolic protein and by bovine serum albumin. Human liver revealed only one 1 alpha-hydroxyvitamin D-3 25-hydroxylase activity located to the heavy mitochondrial fraction. The results are in agreement with previous studies on the localization of vitamin D-3 25-hydroxylase in rat and human liver. The difference in localization of the 25-hydroxylase between rat and human liver implies that studies on the regulation of the microsomal 25-hydroxylase in rat liver may not be relevant to the situation in human liver.     

10-Keto or 25-hydroxy substitution confer equivalent in vitro bone-resorbing activity to vitamin D3

The biological activities of 10-keto derivatives of vitamin D3 and 25-hydroxyvitamin D3 were determined in bone organ culture. Fetal rat limb bones prelabeled with 45Ca were incubated for 60 h with 10-keto-25-hydroxyvitamin D3, 10-keto-vitamin D3, 1,25-dihydroxyvitamin D3, 25-hydroxyvitamin D3, or vitamin D3. Resorption was quantified by release of 45Ca. Substitution of a keto group in the 10 position of the vitamin D3 molecule resulted in a compound equal in potency to 25-hydroxyvitamin D3. When a 10-keto group was substituted in the 25-hydroxy vitamin D3 molecule, the potency was increased 20- to 40-fold. In contrast, 1,25-dihydroxyvitamin D3 was 7500-fold more potent than 25-hydroxyvitamin D3. Since 10-keto-25-hydroxyvitamin D3 has a retention time close to that of 1,25-dihydroxyvitamin D3 on normal-phase HPLC eluted with isopropanol:hexane, it is a possible artifact in the assay of 1,25-dihydroxyvitamin D3. Based upon the observed relative activities of the two compounds, the concentration of 10-keto-25-hydroxyvitamin D3 would have to be greater than 0.8 ng/ml for it to interfere in the bioassay of 1,25-dihydroxyvitamin D3.     

The synthesis and biological activity of 25-hydroxy-26,27-dimethylvitamin D3 and 1,25-dihydroxy-26,27-dimethylvitamin D3: highly potent novel analogs of vitamin D3

We synthesized 25-hydroxy-26,27-dimethylvitamin D3, 9, and 1,25-dihydroxy-26,27-dimethylvitamin D3, 14, from chol-5-enic acid-3 beta-ol and tested their biological activity in vivo and in vitro. 9 was found to be highly potent vitamin D analog with bioactivity similar to that of 25-hydroxyvitamin D3. 9 bound to rat plasma vitamin D binding protein with approximately one-third the affinity of 25-hydroxyvitamin D3. 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 D3. 1,25-Dihydroxy-26,27-dimethylvitamin D3, 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 D3. 14 bound to vitamin D binding protein about 18 times less effectively than 1,25-dihydroxyvitamin D3. 14 bound to the chick intestinal cytosol receptor with an affinity one-half that of 1,25-dihydroxyvitamin D3. In a duodenal organ culture system, 14 was about half as active as 1,25-dihydroxyvitamin D3. 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.     

Stimulatory effect of prostaglandin E2 on 1 alpha,25-dihydroxyvitamin D3 synthesis in rats.

The effect of prostaglandin E2 on accumulation in plasma of 1 alpha,25-dihydroxy[3H]vitamin D3 from 25-hydroxy[3H]vitamin D3 was studied in vivo using vitamin D-deficient thyroparathyroidectomized rats. Intra-arterial infusion of 10-50 micrograms of prostaglandin E2/h caused a significant stimulation of 1 alpha,25-dihydroxy[3H]vitamin D3 production. No significant changes in plasma Ca2+ and Pi concentrations or urinary cyclic AMP excretion were observed after prostaglandin E2 infusion. Theophylline did not enhance the effect of a submaximal dose of prostaglandin E2 on 1 alpha,25-dihydroxy[3H]vitamin D3 production. These data indicate that prostaglandin E2 stimulates plasma accumulation of 1 alpha,25-dihydroxy[3H]vitamin D3 independent of the adenylate cyclase/cyclic AMP system, and suggest that prostaglandin E2 has a modulatory role in the regulation of 25-hydroxyvitamin D3 1 alpha-hydroxylase in the kidney.     

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)).     

Determination of the affinity of vitamin D metabolites to serum vitamin D binding protein using assay employing lipid-coated polystyrene beads.

We have developed an assay to measure the affinity of serum vitamin D binding protein for 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3, and vitamin D3, using uniform diameter (6.4 microns) polystyrene beads coated with phosphatidylcholine and vitamin D metabolites as the vitamin D donor. The lipid metabolite coated beads have a solid core, and thus all of the vitamin D metabolites are on the bead surface from which transfer to protein occurs. After incubating these beads in neutral buffer for 3 h, essentially no 3H-labeled vitamin D metabolites desorb from this surface. Phosphatidylcholine/vitamin D metabolite-coated beads (1 microM vitamin D metabolite) were incubated with varying concentrations of serum vitamin D binding protein under conditions in which the bead surfaces were saturated with protein, but most of the protein was free in solution. After incubation, beads were rapidly centrifuged without disturbing the equilibrium of binding and vitamin D metabolite bound to sDBP in solution was assayed in the supernatant. All three vitamin D metabolites became bound to serum vitamin D binding protein, and after 10 min of incubation the transfer of the metabolites to serum vitamin D binding protein was time independent. The transfer followed a Langmuir isotherm, and the Kd for each metabolite binding to serum vitamin D binding protein was derived by nonlinear least-squares fit analysis. From this analysis the following values for the Kd were obtained: 5.59 x 10(-6) M, 25-hydroxyvitamin D; 9.45 x 10(-6) M, 1,25-dihydroxyvitamin D; and 9.17 x 10(-5) M, vitamin D. In conclusion, we have developed a method which avoids problems encountered in previous assays and allows the precise and convenient determination of binding affinities of vitamin D metabolites and serum vitamin D binding protein.     

25-Hydroxylation of 1-α-hydroxyvitamin D-3 in rat and human liver

1α-Hydroxyvitamin D-3 25-hydroxylase activity was measured in subcellular fractions of rat and human liver. The formation of 1,25-dihydroxyvitamin D-3 was determined by high pressure liquid chromatography. In rat liver 1α-hydroxyvitamin D-3 25-hydroxylase activities were found in the purified nuclei, the heavy mitochondrial fraction and the microsomal fraction. The enrichment of 25-hydroxylase activity was highest in the heavy mitochondrial fraction. With this fraction a minimum amount (about 0.5 mg) of protein was required before formation of 1,25-dihydroxyvitamin D-3 could be detected. Above this amount the reaction was linear with amount of protein up to at least 2 mg/ml. The reaction was also linear with time up to 60 min. An apparent K_m value of 2·10^?5 M was found. The mitochondrial 25-hydroxylase was stimulated by addition of cytosolic protein or bovine serum albumin. The degree of stimulation was dependent on the amount of mitochondrial protein present in the incubation mixture. Maximal stimulation was seen with 0.2 mg/ml of either protein in the presence of 0.5 mg mitochondrial protein. The stimulating effect remained after heating the protein for 5 min at 100°C. The cytosolic protein did not stimulate a reconstituted mitochondrial 1α-hydroxyvitamin D-3 25-hydroxylase. The mitochondrial vitamin D-3 25-hydroxylase was inhibited both by cytosolic protein and by bovine serum albumin. Human liver revealed only one 1α-hydroxyvitamin D-3 25-hydroxylase activity located to the heavy mitochondrial fraction. The results are in agreement with previous studies on the localization of vitamin D-3 25-hydroxylase in rat and human liver. The difference in localization of the 25-hydroxylase between rat and human liver implies that studies on the regulation of the microsomal 25-hydroxylase in rat liver may not be relevant to the situation in human liver.     

Genome-independent effects of 1,25-dihydroxy vitamin D-3 on membrane potential

Cell membrane potential, $\text{V}{}_{\mathrm{<mtext>m</mtext>}}$, was monitored in rabbit hypertrophic cartilage metatarsals, amphibian proximal tubule and muscle cells during application of 1,25-dihydroxy vitamin D-3, 25-hydroxy vitamin D-3 or cholesterol (10^?10M). 1,25-Dihydroxy vitamin D-3 elicited quick variations of $\text{V}{}_{\mathrm{<mtext>m</mtext>}}$ (in less than 1 min) in proximal tubular cells (whether injected in the lumen or in peritubular capillaries) and in cartilage. The precursor 25-hydroxy vitamin D-3 and cholesterol produced a small shift of $\text{V}{}_{\mathrm{<mtext>m</mtext>}}$ in proximal tubule only when applied from the luminal side, but this change was significantly smaller than that observed with 1,25-dihydroxy vitamin D-3. Muscle cells were unresponsive to both metabolites and cholesterol. It is concluded that rapid effects of 1,25-dihydroxy vitamin D-3 on $\text{V}{}_{\mathrm{<mtext>m</mtext>}}$, in target cells, are specific, most likely due to permeability changes and not related to nuclear protein synthesis; they may contribute to early modulation of cell function.     

An improved radioreceptor assay for 1,25-dihydroxyvitamin D in human plasma

We describe a modified assay technique for quantitating 1,25-dihydroxyvitamin D in plasma. The method involves a rapid extraction of the hormone using minicolumn (made of granular diatomaceous earth) chromatography followed by single-step purification on high-performance liquid chromatography. Quantitation of plasma 1,25-dihydroxyvitamin D is achieved by a radioligand receptor assay employing lyophilized cytosolic receptor protein from chick intestine and high-specific-activity 1,25-dihydroxy[³H]vitamin D₃ (166 Ci/mmol). A new incubation medium including an ethanol extract of vitamin D-deficient chick serum yields high specific binding and improves the precision of the radioassay. Bound and free hormone are separated with dextran-coated charcoal of equivalent particle size. The method is sensitive to 0.5 pg/tube with a practical detection range of 1–20 pg/tube, permitting duplicate assay of endogenous 1,25-dihydroxyvitamin D in plasma volumes as small as 0.5 ml. The intra- and interassay coefficient of variation are 5 and 9%, respectively, and the method is valid over a wide-range sample dilution. This assay technique was applied to the measurement of plasma 1,25-dihydroxyvitamin D hormone concentration in normal young adults (55.2 ± 13.6 pg/ml; n = 20) and in patients with chronic renal failure (13.5 ± 5.2 pg/ml; n = 9) and primary hyperparathyroidism (83.3 ± 18 pg/ml; n = 10).