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

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

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)).     

Synthesis, biologic activity, and protein binding characteristics of a new vitamin D analog, 22-hydroxyvitamin D3

We synthesized a novel vitamin D analog, 22-hydroxyvitamin D3 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 D3. The new vitamin 9 was synthesized from 3 beta-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 D3 or 25-hydroxyvitamin D3. It was a weak vitamin D3 agonist in the chick embryonal duodenum in vitro. It did not antagonize the activity of 1,25-dihydroxyvitamin D3. Vitamin 9 bound to the chick intestinal cytosol receptor with low affinity. 22-Hydroxyvitamin D3 and various vitamin D sterols were bound to vitamin D binding protein in the following order: 25-hydroxyvitamin D3. (24R)-24,25-dihydroxyvitamin D3, and (25S)-25,26-dihydroxyvitamin D3 greater than 22-hydroxyvitamin D3 greater than 11 alpha-hydroxyvitamin D3 greater than 1,25-dihydroxyvitamin D3 greater than vitamin D3. We conclude that the introduction of a hydroxyl group at C-22 in the side chain of the vitamin D3 molecule decreases its biological activity.     

Side-chain oxidation of vitamin D3 in mouse kidney mitochondria: effect of the Hyp mutation and 1,25-dihydroxyvitamin D3 treatment

Side-chain oxidation of vitamin D is an important degradative pathway. In the present study we compared the enzymes involved in side-chain oxidation in normal and Hyp mouse kidney. Homogenates of normal mouse kidney catalyze the conversion of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3, 24-oxo-25-hydroxyvitamin D3 and 24-oxo-23,25-dihydroxyvitamin D3. After subcellular fractionation, total side-chain oxidative activity, estimated by the sum of the three products synthesized per milligram protein under initial rate conditions, coincided with the mitochondrial enzyme marker succinate-cytochrome-c reductase. Treatment of normal mice with 1,25-dihydroxyvitamin D3 (1.5 ng/g) resulted in an eightfold increase in mitochondrial enzyme activity, with no change in apparent Km but a significant rise in Vmax. With 24,25-dihydroxyvitamin D3 as the substrate, normal renal mitochondria produced 24-oxo-25-hydroxyvitamin D3 and 24-oxo-23,25-dihydroxyvitamin D3, and the synthesis of these metabolites could be increased sixfold by pretreatment with 1,25-dihydroxyvitamin D3. In the Hyp mouse, the side-chain oxidation pathway showed similar subcellular distribution of enzyme activity. However, product formation from 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 was twofold greater in mutant than in normal mitochondria. Furthermore, 1,25-dihydroxyvitamin D3 pretreatment of Hyp mice resulted in a 3.4-fold increase over basal metabolism of both 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3. These results demonstrate that (i) kidneys from normal and Hyp mice possess basal and 1,25-dihydroxyvitamin D3 inducible enzyme system(s) in the mitochondrial fraction, which catalyze the side-chain oxidation of 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3, and (ii) the Hyp mutation appears to perturb the renal metabolism of both substrates only in the basal state.     

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.     

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.     

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.     

1 alpha-hydroxylation of 24-hydroxyvitamin D2 represents a minor physiological pathway for the activation of vitamin D2 in mammals

C24-Hydroxylation was evaluated as a possible activation pathway for vitamin D2 and vitamin D3. Routine assays showed that 24-hydroxyvitamin D2 and 1,24-dihydroxyvitamin D2 could be detected in rats receiving physiological doses (100 IU/day) of vitamin D2; however, 24-hydroxyvitamin D3 could not be detected in rats receiving similar doses of vitamin D3. In rats, 24-hydroxyvitamin D2 was very similar to 25-hydroxyvitamin D2 at stimulating intestinal calcium transport and bone calcium resorption. The biological activity of 24-hydroxyvitamin D2 was eliminated by nephrectomy, suggesting that 24-hydroxyvitamin D2 must undergo 1 alpha-hydroxylation to be active at physiological doses. In vivo experiments suggested that when given individually to vitamin D deficient rats, 24-hydroxyvitamin D2, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 were 1 alpha-hydroxylated with the same efficiency. However, when presented simultaneously, 24-hydroxyvitamin D2 was less efficiently 1 alpha-hydroxylated than either 25-hydroxyvitamin D3 or 25-hydroxyvitamin D2. 1,24-Dihydroxyvitamin D2 was also approximately 2-fold less competitive than either 1,25-dihydroxyvitamin D2 or 1,25-dihydroxyvitamin D3 for binding sites on the bovine thymus 1,25-dihydroxyvitamin D receptor. These results demonstrate that 24-hydroxylation followed by 1 alpha-hydroxylation of vitamin D2 represents a minor activation pathway for vitamin D2 but not vitamin D3.     

Plasma levels of vitamin D metabolites in fetal and pregnant ewes

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

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.     

Characterization of 25-hydroxyvitamin D binding protein from intestinal cells

We have previously purified a cytosolic vitamin D metabolite binding protein (cDBP) from rat enterocytes, which has characteristics distinct from other vitamin D binding proteins. In these studies, we demonstrate that cDBP in a semi-purified fraction from human intestinal cells (Caco-2 cells) binds 25-hydroxyvitamin D (25OHD) with at least a 1000-fold greater affinity than 1, 25-dihydroxyvitamin D (1,25(OH)(2)D) or 24,25-dihydroxyvitamin D. Treatment of cells with 1,25(OH)(2)D reduced 25OHD binding to approximately one third that of the untreated cells (0.42 CPM/mg total protein vs 1.34 CPM/mg total protein, respectively). Finally, the cDBP is not immunoreactive to antibodies prepared against the C-terminus of the nuclear vitamin D receptor (VDR). In summary, cDBP bound 25OHD with greater affinity than either 1,25(OH)(2)D or 24,25 dihydroxyvitamin D, the cytosolic binding activity was down-regulated by 1,25(OH)(2)D and cBDP is distinct from the nuclear VDR.     

Determination of vitamin D and its metabolites in plasma from normal and anephric man

A multiple assay capable of reliably determining vitamins D(2) and D(3) (ergocalciferol and cholecalciferol), 25(OH)D(2) (25-hydroxyvitamin D(2)) and 25(OH)D(3) (25-hydroxyvitamin D(3)), 24,25(OH)(2)D (24,25-dihydroxyvitamin D), 25,26(OH)(2)D (25,26-dihydroxyvitamin D) and 1,25(OH)(2)D (1,25-dihydroxyvitamin D) in a single 3-5ml sample of human plasma was developed. The procedure involves methanol/methylene chloride extraction of plasma lipids followed by separation of the metabolites and purification from interfering contaminants by batch elution chromatography on Sephadex LH-20 and Lipidex 5000 and by h.p.l.c. (high-pressure liquid chromatography). Vitamins D(2) and D(3) and 25(OH)D(2) and 25(OH)D(3) are quantified by h.p.l.c. by using u.v. detection, comparing their peak heights with those of standards. 24,25(OH)(2)D and 25,26(OH)(2)D are measured by competitive protein-binding assay with diluted plasma from vitamin D-deficient rats. 1,25(OH)(2)D is measured by competitive protein-binding assay with diluted cytosol from vitamin D-deficient chick intestine. Values in normal human plasma samples taken in February are: vitamin D 3.5+/-2.5ng/ml; 25(OH)D 31.6+/-9.3ng/ml; 24,25(OH)(2)D 3.5+/-1.4ng/ml; 25,26(OH)(2)D 0.7+/-0.5ng/ml; 1,25(OH)(2)D 31+/-9pg/ml (means+/-s.d.). Values in two normal human plasma samples taken in February after 1 week of high sun exposure are: vitamin D 27.1+/-7.9ng/ml; 25(OH)D 56.8+/-4.2ng/ml; 24,25(OH)(2)D 4.3+/-1.6ng/ml; 25,26(OH)(2)D 0.5+/-0.2ng/ml. Values in anephric-human plasma are: vitamin D 2.7+/-0.8ng/ml; 25(OH)D 36.4+/-16.5ng/ml; 24,25(OH)(2)D 1.9+/-1.3ng/ml; 25,26(OH)(2)D 0.6+/-0.3ng/ml; 1,25(OH)(2)D was undetectable.     

1,25-Dihydroxyvitamin D3 stimulates 45Ca2+-uptake by cultured vascular smooth muscle cells derived from rat aorta

We have recently shown the presence of receptors for 1,25-dihydroxyvitamin D3 and that 1,25-dihydroxyvitamin D3 stimulates Ca-ATPase in vascular smooth muscle cells presumably via receptor mediated mechanism. These data suggest that the sterol may directly be involved in the regulation of cellular calcium homeostasis. To further define action of vitamin D in smooth muscle cells, we studied effect of the sterol on cellular uptake of calcium. 1,25-dihydroxyvitamin D3 stimulated 45Ca2+ uptake by cultured cells, A7r5, derived from fetal rat aorta, when the cells were incubated with the sterol for 18 hr. The effect was dose-dependent at 10(-10) to 10(-9) M, and three orders of magnitude higher concentration of 25-hydroxyvitamin D3 or 24,25-dihydroxyvitamin D3 was needed to obtain similar effects. Furthermore, the effect of 1,25-dihydroxyvitamin D3 was abolished by cycloheximide (10(-5) M), a protein synthesis inhibitor. These data clearly suggest that 1,25-dihydroxyvitamin D3 may directly regulate cellular calcium homeostasis in vascular smooth muscle cells presumably via receptor mediated mechanism.     

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

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

Regulation of 25-hydroxyvitamin D3 metabolism in a human promyelocytic leukemia cell line (HL-60): 1,25-dihydroxyvitamin D3 stimulates the synthesis of 24,25-dihydroxyvitamin D3

The human promyelocytic leukemia cell line HL-60 undergoes macrophage-like differentiation after exposure to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the biologically active metabolite of vitamin D3. In the current study, we demonstrate that 1,25(OH)2D3 also regulates 25-hydroxyvitamin D3 [25(OH)D3] metabolism in HL-60 cells. The presence of 1,25(OH)2D3 in the culture medium of HL-60 cells stimulated the conversion of 7-10% of the substrate [25(OH)D3] to a more polar metabolite, which was identified as 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] from the elution positions on sequential HPLC systems and the sensitivity to periodate treatment. The HL-60 subclone HL-60 blast, which is unresponsive to 1,25(OH)2D3 in terms of differentiation, also responded to 1,25(OH)2D3 treatment with the production of 24,25(OH)2D3. Maximal stimulation of 24,25(OH)2D3-synthesis (approximately 7 pmol/5 X 10(6) cells) in HL-60 cells was noted with a 12-h exposure to 10(-9) M 1,25(OH)2D3. The ability of vitamin D3 metabolites other than 1,25(OH)2D3 to induce the synthesis of 24,25(OH)2D3 in HL-60 cells was, with the exception of 1 alpha-hydroxyvitamin D3, in correlation with their reported affinities for the specific 1,25(OH)2D3 receptor which is present in HL-60 cells. Treatment of HL-60 cells with phorbol diesters abolished the 1,25(OH)2D3 responsiveness, while treatment with dimethylsulfoxide and interferon gamma did not markedly alter the 25(OH)D3 metabolism of HL-60 cells. Small amounts (approximately 1% of substrate) of two 25(OH)D3 metabolites, which comigrated with 5(E)- and 5(Z)-19-nor-10-keto-25-hydroxyvitamin D3 on two HPLC solvent systems, were synthesized by HL-60 cells, independently from 1,25(OH)2D3 treatment or stage of cell differentiation. Our results indicate that 1,25(OH)2D3 influences 25(OH)D3 metabolism of HL-60 cells independently from its effects on cell differentiation.     

Metabolism of vitamin D3 in the chick embryo

In agreement with previous reports, chick intestinal calcium-binding protein does not appear in the chick embryo until 1 day after hatching while intestinal alkaline phosphatase begins to appear at 19–20 days of embryonic life. The ability of chick embryo to metabolize vitamin D₃ to 25-hydroxyvitamin D₃, 1,25-dihydroxyvitamin D₃, and 24,25-dihydroxyvitamin D₃ is present at least by day 18 of embryonic life as demonstrated by in vivo and in vitro techniques. It also illustrates that metabolism of vitamin D₃ was not the limiting factor in the appearance of calcium-binding protein and alkaline phosphatase in intestine. Instead, the uptake of 1,25-dihydroxyvitamin D₃ by the duodenum was very low prior to hatching, even though significant amounts were present in the yolk sac. Injection of a physiological dose of 1,25-dihydroxyvitamin D₃ to chick embryo at 9 days failed to stimulate appearance of calcium binding protein by 18 days of embryonic life. Thus, it appears that either the normal mechanism for transport of 1,25-dihydroxyvitamin D₃ to intestine or its receptors in intestine may not be present prior to day 18–19.A large fraction of radioactive vitamin D₃ injected into the yolk sac was found esterified especially in the embryonic liver. The significance of this is not yet understood.Injection of 1,25-dihydroxyvitamin D₃ at 325 pmoles/per egg at 9 days resulted in 70% mortality of embryos while a 32-pmole dose resulted in no significant increase in mortality. The basis for this toxicity is not yet understood.     

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

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

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

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

Renal parathyroid hormone-dependent adenylate cyclase in vitamin D-deficient rats. Inhibition by hydroxylated vitamin D3 metabolites

The adenylate cyclase activation by bovine synthetic parathyroid hormone (bPTH) (1-34) was studied in vitro in kidney plasma membranes from D-deficient (D-Mb) or normal (D+Mb) rats. In D-Mb, the apparent affinity of parathyroid hormone (PTH) for membranes (170 +/- 30 nM) was significantly higher than that measured in D+Mb (55 +/- 5 nM). The maximum velocity of the PTH-stimulated adenylate cyclase was significantly higher in D+Mb than in D-Mb (163.0 +/- 13.7 and 93.4 +/- 6.7 pmol of cAMP/mg of protein/min, respectively). The action of vitamin D metabolites on the adenylate cyclase stimulation by PTH was then studied in vitro in D-Mb and D+Mb. In D-Mb, 25-hydroxyvitamin D3, 24,25-, and 1, 25-dihydroxyvitamin D3 significantly inhibited cAMP production in the presence of 0.87 microM of bPTH. Vitamin D3 had no effect. Maximal inhibition (86%) was observed for 1,25-dihydroxyvitamin D3. 1,25-Dihydroxyvitamin D3 decreased the maximum velocity of PTH-stimulated adenylate cyclase but did not modify the bPTH apparent affinity for D-Mb. The vitamin D3 metabolites tested did not modify the cyclase stimulation by isoproterenol, sodium fluoride, or 5'-guanylylimidodiphosphate. The presence of 1,25-dihydroxyvitamin D3 or 25-hydroxyvitamin D3 did not increase the (Na-K)-ATPase or the phosphodiesterase activities. In the presence of 1,25-dihydroxyvitamin D3 and bPTH, the apparent affinity of ATP for the catalytic moiety was not modified. The maximum velocity was decreased. These results suggest an in vitro interaction between hydroxylated vitamin D metabolites and kidney membranes PTH receptor.     

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

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