Opposing actions of methylxanthines and dibutyryl cyclic AMP on 1,25 dihydroxyvitamin D3 production and calcium fluxes in isolated chick renal tubules

In contrast to dibuturyl cyclic AMP, the methylxanthine phosphodiesterase inhibitors theophylline and caffeine were found to inhibit the conversion of 25 hydroxyvitamin D₃ to 1,25 dihydroxyvitamin D₃ in isolated renal tubules from vitamin D deficient chicks. This inhibition occurred at concentrations of methylxanthines which were shown to increase renal tubule cyclic AMP levels. No effect of theophylline or caffeine on 25 hydroxyvitamin D₃ metabolism in isolated chick renal mitochondria was detected. Because of a demonstrated inhibitory action of calcium (10 and 20 μmol/l) on renal mitochondrial conversion of 25 hydroxyvitamin D₃ to 1,25 dihydroxyvitamin D₃, the effect of theophylline and dibutyryl cyclic AMP on cellular calcium-45 efflux and total renal tubule calcium content was estimated. Theophylline 10 mmol/l was found to inhibit renal tubular calcium efflux and to increase total cellular calcium content, while dibutyryl cyclic AMP 1 mmol/l had the reverse effect on both parameters. Divergent actions of the methylxanthines and dibutyryl cyclic AMP on the formation of 1,25 dihydroxyvitamin D₃ and renal tubule calcium efflux and content support the hypothesis that intracellular calcium is an important regulator of renal vitamin D metabolism. The results indicate that observed actions of methylxanthines cannot always be ascribed to cyclic AMP accumulation.     

Biological activity assessment of the 26,23-lactones of 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 and their binding properties to chick intestinal receptor and plasma vitamin D binding protein

The binding of the natural and unnatural diastereoisomers 25-hydroxyvitamin D3-26,23-lactone and 1,25 dihydroxyvitamin D3-26,23-lactone to the vitamin D-binding protein (DBP) and 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] chick intestinal receptor have been investigated. Also, the biological activities, under in vivo conditions, of these compounds, in terms of intestinal calcium absorption (ICA) and bone calcium mobilization (BCM), in the chick are reported. The presence of the lactone ring in the C23-C26 position of the seco-steroid side chain increased two to three times the ability of both 25(OH)D3 and 1,25(OH)2D3 to displace 25(OH)[3H]D3 from the D-binding protein; however, the DBP could not distinguish between the various diastereoisomers. In contrast, the unnatural form (23R,25S) of the 25-hydroxy-lactone was found to be 10-fold more potent than the natural form, and the unnatural (23R,25S)1,25(OH)2D3-26,23-lactone three times more potent than the natural 1,25-dihydroxy-lactone in displacing 1,25(OH)2[3H]D3 from its intestinal receptor. While studying the biological activity of these lactone compounds, it was found that the natural form of the 25-hydroxy-lactone increased the intestinal calcium absorption 48 h after injection (16.25 nmol), while bone calcium mobilization was decreased by the same dose of the 25-hydroxy-lactone. The 1,25-dihydroxyvitamin D3-26,23-lactone in both its natural and unnatural forms was found to be active in stimulating ICA and BCM. These results suggest that the 25-hydroxy-lactone has some biological activity in the chick and that 1,25(OH)2D3-26,23-lactone can mediate ICA and BCM biological responses, probably through an interaction with 1,25-(OH)2D3 specific receptors in these target tissues.     

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

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

1 alpha,25-dihydroxyvitamin D3 stimulates colony formation of chick embryo chondrocytes in soft agar

The effect of vitamin D metabolites on the growth of chick embryo chondrocytes in soft agar was examined. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] at 10(-8)-10(-7) M induced colony formation by chick embryo chondrocytes in soft agar in the presence of 10% fetal bovine serum. Furthermore, 1,25(OH)2D3 increased the number of colonies in the presence of a maximal dose of basic fibroblast growth factor, a potent mitogen for chondrocytes in soft agar. However, 24R,25 (OH)2D3 and other metabolites had little effect on the soft agar growth of chondrocytes in the presence or absence of basic fibroblast growth factor. These results suggest that 1,25(OH)2D3 is an active metabolite which may be involved in supporting cartilage growth.     

Responses of rachitic cartilage cells to metabolites of vitamin D3

Responses of cultured cartilage cells to metabolites of vitamin D3 were studied. Cells were obtained from the epiphyseal growth plate of rachitic chicks and were exposed to physiological and pharmacological concentrations of three metabolites of vitamin D3, 25 hydroxyvitamin D3 (25(OH)D3), 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). 1,25(OH)2D3 was found to reduce L-[U-14C]leucine incorporation into proteins and Na2 35SO4 incorporation into proteoglycans. The synthesis of 24,25(OH)2D3 from 25(OH)D3 was stimulated upon addition of 1,25(OH)2D3 to the cultures. Physiological concentrations of 24,25(OH)2D3 stimulated protein and proteoglycan synthesis. These findings support the notion that vitamin D3, through its active dihydroxylated metabolites, is directly involved in cartilage cells metabolism and healing of rickets.     

6-Fluoro-vitamin D3: a new antagonist of the biological actions of vitamin D3 and its metabolites which interacts with the intestinal receptor for 1 alpha,25(OH)2-vitamin D3

The biological activity and the binding affinity for the 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] intestinal receptor of a new fluorine-containing vitamin D compound, namely 6-fluoro-vitamin D3 (6-F-D3), is reported. A significant interaction of 6-F-D3 with the 1,25(OH)2D3 receptor was found, with a relative competitive index (RCI) of 0.26 +/- 0.04, which is intermediate between 25-hydroxyvitamin D3 (0.14 +/- 0.01) and 1 alpha-hydroxyvitamin D3 (0.46 +/- 0.08), where the RCI of 1,25(OH)2D3 is defined to be 100. In contrast, vitamin D3 was unable to interact with the 1,25(OH)2D3 receptor. Also, the biological activity of 6-F-D3 was assessed in vivo in the vitamin D-deficient chick. 6-F-D3 at doses up to 130 nmol displayed no biological action on either intestinal calcium absorption (ICA) or bone calcium mobilization (BCM) over the time interval of 14-48 h after dosing. However, when 130 nmol 6-F-D3 was given 2 h before and 6 h after vitamin D3 (1.62 nmol), a significant inhibition of vitamin D-mediated ICA was noted. Also, a dose of 130 nmol 6-F-D3 given 2 h before and 6 h after 1,25(OH)2D3 (0.26 nmol) significantly inhibited ICA, as measured at 12 h. 6-F-D3 is the first vitamin D analog found which has an ability to both bind to the 1,25(OH)2D3 receptor and to antagonize the production of biological responses by 1,25(OH)2D3.     

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.     

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.     

A specific binding protein for 1 alpha,25-dihydroxyvitamin D in the chick embryo chorioallantoic membrane

A 3.7 S binding protein for the steroid hormone and vitamin D metabolite 1 alpha-25-dihydroxyvitamin D (1,25-(OH)2-D) was observed in high salt cytosol extracts of chick embryo chorioallantoic membrane. The binding protein was characterized after partial purification of cytosol extracts by ammonium sulfate fractionation. The binding of 1,25-(OH)2-D was saturable, had a high affinity (Kd = 0.16 nM), and was specific for hormonally active vitamin D metabolites. Analysis of the displacement of [3H]1,25-(OH)2-D by unlabeled analogues showed the affinities of vitamin D metabolites to be in the order of 1,25-(OH)2-D = 1,24R,25-(OH)3-D much greater than 25-OH-D = 1-OH-D greater than 24R,25-(OH)2-D. Hormone binding was sensitive to pretreatment with sulfhydryl-blocking reagents. The chorioallantoic membrane 1,25-(OH)2-D-binding protein associated with the chromatin fraction after homogenization of membranes in low salt buffer, and bound to DNA-cellulose columns, eluting as a single peak at 0.215 M KCl. These findings support identification of this 1,25-(OH)2-D-binding protein as a steroid hormone receptor, with properties indistinguishable from 1,25-(OH)2-D receptors in other chick tissues. The chorioallantoic membrane functions in the last third of embryonic development to reabsorb calcium from the eff shell for deposition in embryonic bone. 1,25-(OH)2-D binding activity in the chorioallantoic membrane increased 4- to 5-fold from day 12 to day 16 of incubation, immediately preceding the onset of shell reabsorption. This finding suggests that 1,25-(OH)2-D may act to regulate shell mobilization and transepithelial calcium transport by the chorioallantoic membrane. Finally, the similarity of shell mobilization to bone resorption, which is also stimulated by 1,25-(OH)2-D, suggests that the chorioallantoic membrane is a useful alternate model for the study of 1,25-(OH)2-D action on bone mineral metabolism.     

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)).     

Early effects of 1,25 dihydroxyvitamin D on bone calcium in vitamin D-deficient rats

The early effects of 1,25 dihydroxyvitamin D [1,25 (OH)2D] on calcium transfer in and out of the skeleton were studied in rats to determine whether mobilized bone calcium was reutilized during new bone mineralization. Vitamin-D deficient rats were labeled with 45calcium 10 to 14 days prior to treatment (experiment 1) or at the same time (experiment 2) they were injected with 0.125 microgram of 1,25 (OH)2D. Blood and bone samples were collected from 30 min to 24 h following 1,25 (OH)2D injection. Stable and radioactive calcium were determined in serum, and caudal vertebrae were subjected to histomorphometric and autoradiographic studies. In the rats of experiment 1, serum specific radioactivity peaked from 1 to 3 h after 1,25 (OH)2D injection, while there was no change in control rats receiving the vehicle alone. In the untreated vitamin D-deficient rats of experiment 2, the rate of 45calcium loss in serum was higher than normal but returned to normal after 1,25 (OH)2D injection. Serum calcium and osteoclast number remained initially unchanged, suggesting that 1,25 (OH)2D acted by increasing the efflux of calcium from bone and/or by stimulating the activity of existing osteoclasts. The rapid mobilization of 45calcium, accompanied by an increase in the extent of actively mineralizing surfaces, was followed by an increase in the extent of endosteal surface with osteoblasts and by specific incorporation of radioactive calcium at sites of new bone calcification. This study indicates that in vitamin D-deficient rats, the initial promotion of bone mineralization by 1,25 (OH)2D resulted in part from the rapid mobilization of calcium from old mineralized bone.     

Cultured human keratinocytes cannot metabolize vitamin D3 to 25-hydroxyvitamin D3.

The metabolism of [3H]vitamin D3 was studied in cultured human keratinocytes (CHK). Intact CHK were incubated for 1, 6, 12, 24 and 48 h with [3H]vitamin D3 and the lipid soluble fractions from the media and cells were extracted by high-performance liquid chromatography (HPLC). Vitamin D3 and its metabolites, 25-OH-D3, 24,25(OH)2D3 and 1,25(OH)2D3 were added to the extracts, as markers, prior to HPLC. HPLC analysis of the lipid extracts did not reveal any monohydroxylated metabolites. CHK incubated for one hour with [3H]25-OH-D3 showed a 10 +/- 4% conversion to [3H]1,25(OH)2D3 whereas no conversion to [3H]1,25(OH)2D3 was observed in control CHKs that were boiled prior to incubation with [3H]25-OH-D3. These findings suggest that cultured neonatal keratinocytes are incapable of metabolizing vitamin D3 to 25-OH-D3.     

Action of 1,25-dihydroxyvitamin D3 and parathyroid hormone on 45calcium uptake by the yolk sac membrane of chick embryos

During development, the chick embryo mobilizes the calcium it needs from two extraembryonic sources, initially from the yolk and later from the eggshell. Calcium may be hormonally regulated during avian embryogenesis, but details of this regulation are lacking. We investigated the effects of 1,25-dihydroxycholecalciferol [1,25(OH)2D3], bovine parathyroid hormone [bPTH], and vehicle [ethanol or saline] on blood calcium values and incorporation of 45Ca into the yolk sac membrane of 9, 12, and 15 day chick embryos. Control data were also collected from uninjected 6 day embryos. Solutions were injected directly into the yolk sac compartment 48 and 24 hours prior to the experiment. Exogenous 1,25(OH)2D3 induced hypercalcemia in all age groups examined, while exogenous PTH induced hypercalcemia in day 12 and 15 embryos. Small disks of yolk sac membrane were incubated in medium to which 45Ca was added and assayed for 45Ca content at various intervals after start of incubation. In control yolk sac tissue, the uptake of 45Ca was greatest in younger embryos with decreasing uptake at developmentally more advanced ages; 1,25(OH)2D3 treatment significantly enhanced the uptake of 45Ca into yolk sac tissue in all groups (9, 12, and 15 day embryos). PTH treatment caused a significant elevation in 45Ca uptake in the day 12 and 15 embryos.     

The appearance of 1,25-dihydroxyvitamin D3 receptor during chick embryo development

The appearance of the 1,25-dihydroxyvitamin D3 receptor in intestine, kidney, and chorioallantoic membrane of chick embryo was followed by sucrose density gradient sedimentation analysis and Scatchard plot analysis. The receptor from each of these organs sediments as a single 3.7S component. At 19 days of embryonic life, intestine had the highest specific 1,25-dihydroxyvitamin D3 binding activity followed by kidney and chorioallantoic membrane. The 1,25-dihydroxyvitamin D3 binding activity increased gradually at 12-15 days and rapidly until 20 days in intestine. In kidney, this protein increased rapidly from 12 to 16 days and did not change subsequently. In chorioallantoic membrane, the receptor increased slowly from 8 through 15 days, rapidly until 19 days, and decreased at 20 days. The injection of hydrocortisone into the chick embryo at 10 days increased receptor number in intestine, kidney, and chorioallantoic membrane by a factor of 2 at 12 days. Injection of this hormone after this time had little or no effect.     

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).     

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.     

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.     

The vitamin D metabolome: An update on analysis and function

Current understanding of vitamin D tends to be focussed on the measurement of the major circulating form 25‐hydroxyvitamin D3 (25OHD3) and its conversion to the active hormonal form, 1α,25‐dihydroxyvitamin D3 (1α,25(OH)₂D3) via the enzyme 25‐hydroxyvitamin D‐1α‐hydroxylase (CYP27B1). However, whilst these metabolites form the endocrine backbone of vitamin D physiology, it is important to recognise that there are other metabolic and catabolic pathways that are now recognised as being crucially important to vitamin D function. These pathways include C3‐epimerization, CYP24A1 hydroxylase, CYP11A1 alternative metabolism of vitamin D3, and phase II metabolism. Endogenous metabolites beyond 25OHD3 are usually present at low endogenous levels and may only be functional in specific target tissues rather than in the general circulation. However, the technologies available to measure these metabolites have also improved, so that measurement of alternative vitamin D metabolic pathways may become more routine in the near future. The aim of this review is to provide a comprehensive overview of the various pathways of vitamin D metabolism, as well as describe the analytical techniques currently available to measure these vitamin D metabolites.     

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.