25-Hydroxyvitamin D and 24,25-dihydroxyvitamin D in maternal plasma,fetal plasma and amniotic fluid in the rat

At the end of gestation plasma levels of 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D were lower in pregnant than non pregnant female rats. In fetal plasma, concentrations of both metabolites were higher than in maternal plasma. This materno-fetal gradient led us to compare maternal and fetal plasma binding abilities. Fetal plasma was half as potent in binding 25-hydroxyvitamin D as maternal plasma. In fetal plasma binding was mainly due to the plasma vitamin D binding protein. On the other hand this study clearly showed that amniotic fluid contained 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D. In addition this fluid was found to possess vitamin D-metabolite binding activity. The molecule responsible for this has been identified as the plasma vitamin D binding protein.     

Acid-catalyzed esterification of lysozyme in methanol. Reactivities of individual carboxyl groups

Rats maintained on a diet low in phosphorus produce 1,25-dihydroxyvitamin D₃ from 25-hydroxyvitamin D₃ whether they have been thyroparathyroidectomized or not. On the other hand, rats maintained on low-calcium diets produce 1,25-dihydroxyvitamin D₃, but lose this ability within 48 hr after thyroparathyroidectomy. This loss of ability to synthesize 1,25-dihydroxyvitamin D₃ can be prevented or be restored by replacing their drinking water with calcium gluconate-glucose solution which returns their high serum inorganic phosphorus to normal levels. In thyroparathyroidectomized rats under a variety of conditions, the ability to synthesize 1,25-dihydroxyvitamin D₃ correlates with serum inorganic phosphorus values below 7–8 mg/100 ml while the ability to synthesize 24,25-dihydroxyvitamin D₃ correlates with serum phosphorus values above 7–8 mg/100 ml. There is in addition a close correlation between reduced kidney cortex inorganic phosphorus levels and the synthesis of 1,25-dihydroxyvitamin D₃. It is suggested that the renal tubular cell inorganic phosphorus level underlies the regulation of synthesis of 1,25-dihydroxyvitamin D₃ in the kidney and that the parathyroid hormone and calcitonin regulate 1,25-dihydroxyvitamin D₃ synthesis via their effects on renal cell inorganic phosphorus levels.     

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)).     

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.     

Stimulation of 1,25-dihydroxyvitamin D3 production by 1,25-dihydroxyvitamin D3 in the hypocalcaemic rat.

Serum 1,25-dihydroxyvitamin D3 concentration and renal 25-hydroxyvitamin D 1 alpha-hydroxylase activity were measured in rats fed various levels of calcium, phosphorus and vitamin D3. Both calcium deprivation and phosphorus deprivation greatly increased circulating levels of 1,25-dihydroxyvitamin D3. The circulating level of 1,25-dihydroxyvitamin D3 in rats on a low-calcium diet increased with increasing doses of vitamin D3, whereas it did not change in rats on a low-phosphorus diet given increasing doses of vitamin D3. In concert with these results, the 25-hydroxyvitamin D 1 alpha-hydroxylase activity was markedly increased by vitamin D3 administration to rats on a low-calcium diet, whereas the same treatment of rats on a low-phosphorus diet had no effect and actually suppressed the 1 alpha-hydroxylase in rats fed an adequate-calcium/adequate-phosphorus diet. The administration of 1,25-dihydroxyvitamin D3 to vitamin D-deficient rats on a low-calcium diet also increased the renal 25-hydroxy-vitamin D 1 alpha-hydroxylase activity. These results demonstrate that the regulatory action of 1,25-dihydroxyvitamin D3 on the renal 25-hydroxyvitamin D3 1 alpha-hydroxylase is complex and not simply a suppressant of this system.     

Support of embryonic chick survival by vitamin D metabolites

The provision of 1,25-dihydroxyvitamin D3 as the only source of dietary vitamin D3 to laying hens failed to support normal embryonic development in their fertile eggs. Significant (P less than .001) improvement in embryonic survival to hatching in these eggs resulted from injections of 1,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin D3, 25-hydroxyvitamin D3, or 24,24-difluoro-25-hydroxyvitamin D3 prior to incubation. Maximum embryonic survival with lowest embryonic mortality was observed when 0.20 micrograms/egg of 1,25-dihydroxyvitamin D3 or 0.60 micrograms/egg 25-hydroxyvitamin D3 was injected. These results indicate that several forms of vitamin D, two of which cannot be converted to 24,25-dihydroxyvitamin D3, can provide this activity; and of the vitamin D compounds tested, 1,25-dihydroxyvitamin D3 may be the most active in supporting embryonic survival in the chick when delivered directly by injection.     

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.     

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.     

Effect of dietary calcium and phosphorus on intestinal calcium absorption and vitamin D metabolism.

To understand better dietary regulation of intestinal calcium absorption, a quantitative assessment of the metabolites in plasma and duodenum of rats given daily doses of radioactive vitamin D₃ and diets differing in calcium and phosphorus content was made. All known vitamin D metabolites were ultimately identified by high-pressure liquid chromatography. In addition to the known metabolites (25-hydroxyvitamin D₃, 24,25-dihydroxyvitamin D₃, 1,25-dihydroxyvitamin D₃, 25,26-dihydroxyvitamin D₃, and 1,24,25-trihydroxyvitamin D₃), several new and unidentified metabolites were found. In addition to 1,25-dihydroxyvitamin D₃ and 1,24,25-trihydroxyvitamin D₃, the levels of some of the unknown metabolites could be correlated with intestinal calcium transport. However, whether or not any of these metabolites plays a role in the stimulation of intestinal calcium absorption by low dietary calcium or low dietary phosphorus remains unknown.     

Interrelations of calcium-regulating hormones during normal pregnancy.

Profound changes in calcium metabolism occur during pregnancy. The mother has to make available extra calcium for fetal requirements while ensuring that her plasma and bone calcium concentrations are satisfactorily maintained. In a cross-sectional study plasma concentrations of the major calcium-regulating hormones--namely, calcitonin, parathyroid hormone, 25-hydroxyvitamin D (25-OHD), and 1,25-dihydroxyvitamin D (1,25-(OH)2D)--were measured to establish their interrelations during normal pregnancy. The major changes observed were increases in the circulating concentrations of 1,25-(OH)2D and calcitonin. Concentrations of parathyroid hormone and 25-OHD remained within the normal range. The increased concentrations of 1,25-(OH)2D enable the increased physiological need for calcium to be met by enhancing intestinal absorption of this element. The simultaneous rise in calcitonin opposes the bone-resorbing activities of 1,25-(OH)2D, thereby protecting the integrity of the maternal skeleton. Maternal calcium homeostasis is thus maintained yet the requirements of the fetus are fulfilled.     

Altered Calcium and Vitamin D Homeostasis in First-Time Calcium Kidney Stone-Formers

Background

Elevated serum 1,25-dihydroxyvitamin D (1,25(OH)₂D) concentrations have been reported among cohorts of recurrent calcium (Ca) kidney stone-formers and implicated in the pathogenesis of hypercalciuria. Variations in Ca and vitamin D metabolism, and excretion of urinary solutes among first-time male and female Ca stone-formers in the community, however, have not been defined.

Methods

In a 4-year community-based study we measured serum Ca, phosphorus (P), 25-hydroxyvitamin D (25(OH)D), 1,25(OH)₂D, 24,25-dihydroxyvitamin D (24,25(OH)₂D), parathyroid hormone (PTH), and fibroblast growth factor-23 (FGF-23) concentrations in first-time Ca stone-formers and age- and gender frequency-matched controls.

Results

Serum Ca and 1,25(OH)₂D were increased in Ca stone-formers compared to controls (P = 0.01 and P = 0.001). Stone-formers had a lower serum 24,25(OH)₂D/25(OH)D ratio compared to controls (P = 0.008). Serum PTH and FGF-23 concentrations were similar in the groups. Urine Ca excretion was similar in the two groups (P = 0.82). In controls, positive associations between serum 25(OH)D and 24,25(OH)₂D, FGF-23 and fractional phosphate excretion, and negative associations between serum Ca and PTH, and FGF-23 and 1,25(OH)₂D were observed. In SF associations between FGF-23 and fractional phosphate excretion, and FGF-23 and 1,25(OH)₂D, were not observed. 1,25(OH)₂D concentrations associated more weakly with FGF-23 in SF compared with C (P <0.05).

Conclusions

Quantitative differences in serum Ca and 1,25(OH)₂D and reductions in 24-hydroxylation of vitamin D metabolites are present in first-time SF and might contribute to first-time stone risk.     

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.     

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.     

The vitamin D endocrine system and bone tissue mineral metabolism in rats with adjuvant arthritis: the effect of 1,25-dihydroxyvitamin D3]

Adjuvant arthritis was induced in male rats by injecting bacillus Calmette-Guèrin in mineral oil in a hindpaw. A decrease in bone density, calcium and phosphorus content due to polyarthritis was found in the tibia of the noninjected hind leg. Arthritic rats demonstrated serum 1,25-dihydroxyvitamin D deficiency along with constant level of 25-hydroxyvitamin D. The disease caused a significant expression of 1,25-dihydroxyvitamin D3 receptors in lymphocytes. Arthritic rats were treated with 1,25-dihydroxyvitamin D3 (0.15 mg/kg/day orally) for 35 days. The treatment prevented the development of osteoporosis and a decrease of 1,25-dihydroxyvitamin D levels as well as reduced the expression of 1,25-dihydroxyvitamin D receptors in lymphocytes.     

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).     

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.     

Stimulation of 25-hydroxyvitamin D3-1alpha-hydroxylase by phosphate depletion.

The ability of low phosphorus diets to stimulate the activity of the 25-hydroxyvitamin D3-1alpha-hydroxylase was tested in the chick. Feeding low phosphorus diets for 2 weeks resulted in a marked increase in enzyme activity relative to chicks fed a normal phosphorus diet. Stimulation of the 25-hydroxyvitamin D3-1alpha-hydroxylase activity by low phosphorus diets, however, was not as great as that observed with a low calcium diet. The low phosphorus and low calcium diets probably results from increased 1,25-dihydroxyvitamin D3 synthesis, whereas the stimulation by phosphate deprivation is only partly the result of increased 1,25-dihydroxyvitamin D3 production.     

Kinetic properties of 25-hydroxyvitamin D- and 1,25-dihydroxyvitamin D-24-hydroxylase from chick kidney

1,25-Dihydroxyvitamin D3 induces both 25-hydroxyvitamin D3- and 1,25-dihydroxyvitamin D3- 24-hydroxylase activities. However, whether 24-hydroxylation of these substrates is catalyzed by a single enzyme is unknown. We have examined the substrate specificity of the enzyme using the solubilized and reconstituted chick renal mitochondrial 24-hydroxylase enzyme system. The soluble enzyme catalyzes 24-hydroxylation of both substrates. The apparent Km of the 24-hydroxylase for 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 were 1.47 and 0.14 microM, respectively. Kinetic studies demonstrated that 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 act as competitive inhibitors with respect to each other. 1,25-Dihydroxyvitamin D3 inhibited the production of 24,25-dihydroxyvitamin D3 with an apparent Ki of 0.09 microM and 25-hydroxyvitamin D3 inhibited the production of 1,24,25-trihydroxyvitamin D3 with an apparent Ki of 3.9 microM. These results indicate that chick 24-hydroxylase preferentially hydroxylates 1,25-dihydroxyvitamin D3 and support the idea that the 24-hydroxylation of these substrates is catalyzed by a single enzyme.     

Biological activities and binding properties of 23,23-difluoro-25-hydroxyvitamin D3 and its 1 alpha-hydroxy derivative

23,23-Difluoro-25-hydroxyvitamin D3 is 5-10 times less active than 25-hydroxyvitamin D3 in stimulating intestinal calcium transport, bone calcium mobilization, increasing serum phosphorus, mineralization of rachitic bone, and binding to the plasma transport protein in rats. It is converted to 23,23-difluoro-1 alpha, 25-dihydroxyvitamin D3 by chick renal 25-hydroxyvitamin D-1-hydroxylase. This compound is one-seventh as active as 1,25-dihydroxyvitamin D3 in binding to the chick intestinal receptor for 1,25-dihydroxyvitamin D3. Thus, fluoro substitution on carbon-23 of vitamin D has an unexpected and unexplained suppressive action on plasma binding and biological activity. However, since this substitution does not block the biological response of 25-hydroxyvitamin D3, these results provide additional evidence that 23-hydroxylation of vitamin D is not involved in biological function.