1,25-Dihydroxyvitamin D3 and 20-Hydroxyvitamin D3 Upregulate LAIR-1 and Attenuate Collagen Induced Arthritis.

Vitamin D plays a crucial role in regulation of the immune response. However, treatment of autoimmune diseases with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] doses sufficient to be effective is prohibitive due to its calcemic and toxic effects. We use the collagen-induced arthritis (CIA) model to analyze the efficacy of the noncalcemic analog of vitamin D, 20S-hydroxyvitamin D3 [20S(OH)D3], as well as 1,25(OH)2D3, to attenuate arthritis and explore a potential mechanism of action. Mice fed a diet deficient in vitamin D developed a more severe arthritis characterized by enhanced secretion of T cell inflammatory cytokines, compared to mice fed a normal diet. The T cell inflammatory cytokines were effectively suppressed, however, by culture of the cells with 20S(OH)D3. Interestingly, one of the consequences of culture with 1,25(OH)2D3 or 20S(OH)D3, was upregulation of the natural inhibitory receptor leukocyte associated immunoglobulin-like receptor-1 (LAIR-1 or CD305). Polyclonal antibodies which activate LAIR-1 were also capable of attenuating arthritis. Moreover, oral therapy with active forms of vitamin D suppressed arthritis in LAIR-1 sufficient DR1 mice, but were ineffective in LAIR-1-/- deficient mice. Taken together, these data show that the effect of vitamin D on inflammation is at least, in part, mediated by LAIR-1 and that non-calcemic 20S(OH)D3 may be a promising therapeutic agent for the treatment of autoimmune diseases such as Rheumatoid Arthritis.

20S-Hydroxyvitamin D3, a Secosteroid Produced in Humans, Is Anti-Inflammatory and Inhibits Murine Autoimmune Arthritis.

The ability to use large doses of vitamin D3 (D3) to chronically treat autoimmune diseases such as rheumatoid arthritis (RA) is prohibitive due to its calcemic effect which can damage vital organs. Cytochrome P450scc (CYP11A1) is able to convert D3 into the noncalcemic analog 20S-hydroxyvitamin D3 [20S(OH)D3]. We demonstrate that 20S(OH)D3 markedly suppresses clinical signs of arthritis and joint damage in a mouse model of RA. Furthermore, treatment with 20S(OH)D3 reduces lymphocyte subsets such as CD4+ T cells and CD19+ B cells leading to a significant reduction in inflammatory cytokines. The ratio of T reg cells (CD4+CD25+Foxp3+ T cells) to CD3+CD4+ T cells is increased while there is a decrease in critical complement-fixing anti-CII antibodies. Since pro-inflammatory cytokines and antibodies against type II collagen ordinarily lead to destruction of cartilage and bone, their decline explains why arthritis is attenuated by 20(OH) D3. These results provide a basis for further consideration of 20S(OH)D3 as a potential treatment for RA and other autoimmune disorders.

Validation and Determination of 25(OH) Vitamin D and 3-Epi25(OH)D3 in Breastmilk and Maternal- and Infant Plasma during Breastfeeding.

Vitamin D deficiency in pregnant women and their offspring may result in unfavorable health outcomes for both mother and infant. A 25hydroxyvitamin D (25(OH)D) level of at least 75 nmol/L is recommended by the Endocrine Society. Validated, automated sample preparation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were used to determine the vitamin D metabolites status in mother-infant pairs. Detection of 3-Epi25(OH)D3 prevented overestimation of 25(OH)D3 and misclassification of vitamin D status. Sixty-three percent of maternal 25(OH)D plasma levels were less than the recommended level of 25(OH)D at 3 months. Additionally, breastmilk levels of 25(OH)D decreased from 60.1 nmol/L to 50.0 nmol/L between six weeks and three months (p < 0.01). Furthermore, there was a positive correlation between mother and infant plasma levels (p < 0.01, r = 0.56) at 3 months. Accordingly, 31% of the infants were categorized as vitamin D deficient (25(OH)D < 50 nmol/L) compared to 25% if 3-Epi25(OH)D3 was not distinguished from 25(OH)D3. This study highlights the importance of accurate quantification of 25(OH)D. Monitoring vitamin D metabolites in infant, maternal plasma, and breastmilk may be needed to ensure adequate levels in both mother and infant in the first 6 months of infant life.

Vitamin D and Its Synthetic Analogs.

For many individuals, in particular during winter, supplementation with the secosteroid vitamin D3 is essential for the prevention of bone disorders, muscle weakness, autoimmune diseases, and possibly also different types of cancer. Vitamin D3 acts via its metabolite 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] as potent agonist of the transcription factor vitamin D receptor (VDR). Thus, vitamin D directly affects chromatin structure and gene regulation at thousands of genomic loci, i.e., the epigenome and transcriptome of its target tissues. Modifications of 1,25(OH)2D3 at its side-chain, A-ring, triene system, or C-ring, alone and in combination, as well as nonsteroidal mimics provided numerous potent VDR agonists and some antagonists. The nearly 150 crystal structures of VDR's ligand-binding domain with various vitamin D compounds allow a detailed molecular understanding of their action. This review discusses the most important vitamin D analogs presented during the past 10 years and molecular insight derived from new structural information on the VDR protein.

Differential antitumor effects of vitamin D analogues on colorectal carcinoma in culture.

Colorectal cancer (CRC) is an emerging global problem with the rapid increase in its incidence being associated with an unhealthy lifestyle. Epidemiological studies have shown that decreased levels of vitamin D3 significantly increases the risk of CRC. Furthermore, negative effects of vitamin D3 deficiency can be compensated by appropriate supplementation. Vitamin D3 was shown to inhibit growth and induce differentiation of cancer cells, however, excessive vitamin D3 intake leads to hypercalcemia. Thus, development of efficient vitamin D3 analogues with limited impact on calcium homeostasis is an important scientific and clinically relevant task. The aims of the present study were to compare the antiproliferative potential of classic vitamin D3 metabolites (1α,25(OH)2D3 and 25(OH)D3) with selected low calcemic analogues (calcipotriol and 20(OH)D3) on CRC cell lines and to investigate the expression of vitamin D-related genes in CRC cell lines and clinical samples. Vitamin D3 analogues exerted anti-proliferative effects on all CRC cell lines tested. Calcipotriol proved to be as potent as 1α,25(OH)2D3 and had more efficacy than 20-hydroxyvitamin D3. In addition, the analogs tested effectively inhibited the formation of colonies in Matrigel. The expression of genes involved in 1α,25(OH)2D3 signaling and metabolism varied in cell lines analysed, which explains in part their different sensitivities to the various analogues. In CRC biopsies, there was decreased VDR expression in tumor samples in comparison to the surgical margin and healthy colon samples (p<0.01). The present study indicates that vitamin D3 analogues which have low calcemic activity, such as calcipotriol or 20(OH)D3, are very promising candidates for CRC therapy. Moreover, expression profiling of vitamin D-related genes is likely to be a powerful tool in the planning of anticancer therapy. Decreased levels of VDR and increased CYP24A1 expression in clinical samples underline the importance of deregulation of vitamin D pathways in the development of CRC.

The 3 epimer of 25-hydroxycholecalciferol is present in the circulation of the majority of adults in a nationally representative sample and has endogenous origins.

Fundamental knowledge gaps in relation to the 3 epimer of 25-hydroxycholecalciferol [3-epi-25(OH)D3] limit our understanding of its relevance for vitamin D nutrition and health. The aims of this study were to characterize the 3-epi-25(OH)D3 concentrations in a nationally representative sample of adults and explore its determinants. We also used data from a recent randomized controlled trial (RCT) of supplemental cholecalciferol (vitamin D3) conducted in winter in older adults to directly test the impact of changes in vitamin D status on serum 3-epi-25(OH)D3 concentrations. Serum 25-hydroxycholecalciferol [25(OH)D3] and 3-epi-25(OH)D3 concentrations (via LC-tandem mass spectrometry) from our vitamin D3 RCT in adults (aged ≥50 y) and data on dietary, lifestyle, and biochemical characteristics of participants of the recent National Adult Nutrition Survey in Ireland (aged 18-84 y; n = 1122) were used in the present work. In the subsample of participants who had serum 3-epi-25(OH)D3 concentrations greater than the limit of quantification (n = 1082; 96.4%), the mean, 10th, 50th (median), and 90th percentile concentrations were 2.50, 1.05, 2.18, and 4.30 nmol/L, respectively, whereas the maximum 3-epi-25(OH)D3 concentration was 15.0 nmol/L. A regression model [explaining 29.9% of the variability in serum 3-epi-25(OH)D3] showed that age >50 y, vitamin D supplement use, dietary vitamin D, meat intake, season of blood sampling, and sun exposure habits were significant positive determinants, whereas increasing waist circumference and serum 25-hydroxyergocalciferol concentration were significant negative determinants. The RCT data showed that mean serum 25(OH)D3 and 3-epi-25(OH)D3 concentrations increased (49.3% and 42.1%, respectively) and decreased (-28.0% and -29.1%, respectively) significantly (P < 0.0001) with vitamin D3 (20 µg/d) and placebo supplementation, respectively, over 15 wk of winter. In conclusion, we provide data on serum 3-epi-25(OH)D3 in a nationally representative sample of adults. Our combined observational and RCT data might suggest that both dietary supply and dermal synthesis of vitamin D3 contribute to serum 3-epi-25(OH)D3 concentration.

Analytical measurement and clinical relevance of vitamin D(3) C3-epimer.

With an ever-increasing clinical interest in vitamin D insufficiency, numerous automated immunoassays, protein binding assays, and in-house LC-MS/MS methods are being developed for the quantification of 25-hydroxyvitamin D(3) (25(OH)D(3)). Recently, LC-MS/MS methods have identified an epimeric form of 25(OH)D(3) that has been shown to contribute significantly to 25(OH)D(3) concentration, particularly in infant populations. This review describes the metabolic pathway and physiological functions of 3-epi-vitamin D, compares the capability of various 25(OH)D(3) methods to detect the epimer, and highlights recent publications quantifying 3-epi-25(OH)D(3) in infant, pediatric, and adult populations. In total, this review summarizes the information necessary for clinicians and laboratorians to decide whether or not to report/consider the C3-epimer in the analysis and clinical assessment of vitamin D status.

Serum levels of 3-epi-25-OH-D3 during hypervitaminosis D in clinical practice.

CONTEXT: Intoxication from vitamin D supplements has been rarely reported, but nowadays, it occurs more frequently. The presence of the C-3 epimer of 25-hydroxyvitamin D(3) (3-epi-25-OH-D(3)) is highly prevalent in adults, although there is little information regarding its in vivo relevance, if any, especially under pathological conditions. OBJECTIVE: Our aim was to assess the presence of the 3-epi-25-OH-D(3) in serum samples displaying 25-OH-D(3) concentrations indicative of hypervitaminosis D. DESIGN, SETTING, PATIENTS, AND MAIN OUTCOME MEASURE: A total of 58 samples displaying a wide range of concentrations of 25-OH-D(3) (>64-439 ng/ml) by ultrafast liquid chromatography were consecutively recruited and reassessed for the presence of 3-epi-25-OH-D(3) using a second chromatographic system. Data from additional biochemical tests performed as part of the patient evaluation were also recorded. RESULTS: Mean relative contribution of 3-epi-25-OH-D(3) was less than 4%, and concentrations ranged from 2-28.6 ng/ml. Serum levels of the C3 epimer, but not the relative contribution, correlate with serum 25-OH-D(3). Overall, in subjects with 25-OH-D(3) concentrations indicative of hypervitaminosis D, the presence of the C-3 epimer and its levels were apparently unrelated to age, serum markers of renal and liver function, acute-phase reactants, and the presence of hypercalcemia. 3-Epi-25-OH-D(3) did not correlate with PTH, but subjects displaying PTH suppression (<14 pg/ml) showed higher concentrations of 3-epi-25-OH-D(3). CONCLUSION: The relative contribution of 3-epi-25-D(3) was not significantly altered during hypervitaminosis D, although the absolute levels reached in serum may be biologically relevant. From a clinical viewpoint, although the small size of the group may affect the lack of relationships, the presence of 3-epi-25-OH-D(3) was apparently unrelated to serum markers of renal and liver function, acute-phase reactants, PTH, and the presence of hypercalcemia.

Inhibition of prostate cancer-meditated osteoblastic bone lesions by the low-calcemic analog 1alpha-hydroxymethyl-16-ene-26,27-bishomo-25-hydroxy vitamin D3.

Prostate cancer metastasizes almost exclusively into the bone whereby it induces primarily an osteoblastic response. Non-calcemic vitamin D analogs have been shown to inhibit proliferation of prostate cancer cells in culture and inhibit their growth as subcutaneous xenografts in mice. However, their effect on prostate cancer cell growth in the bone has not been examined. In the present study, we inoculated the osteoblastic prostate cancer cell line MDA-PCa 2b into the bone of male SCID mice and examined the effect of the low-calcemic hybrid analog 1alpha-hydroxymethyl-16-ene-26,27-bishomo-25-hydroxy vitamin D(3) (JK-1626-2) on their ability to induce bone lesions. We found that 7 weeks after inoculation of MDA-PCa 2b cells, 90% of the mice in the vehicle-treated group had significant bone lesions that were detectable by micro-computed tomography and characterized by thickening of the cortical bone and ossification of the epiphysis. Only 30% of the mice in the analog-treated group (daily injections of 4microg/kg, 5 days/week for up to 7 weeks) had detectable bone lesions. Histological examination of the decalcified tumor-bearing bones has shown that tumor cells completely replaced the bone marrow in the diaphysis, and destroyed the trabecular bone in the metaphysis in 90% of the vehicle-treated mice. In contrast, the metaphysis of 60% of analog-treated mice appeared normal, although tumor cells were still found in the diaphysis of 70% of the bones in the analog-treated group. There was no evidence of hypercalcemia in any of the analog-treated mice. In a co-culture, MDA-PCa 2b cells induced a profound mitogenic response in osteoblasts followed by enhanced differentiation. However, in the presence of the analog the mitogenic response of the osteoblasts to the malignant cells was significantly attenuated. These experiments led to the hypothesis that, in vivo, JK-1626-2 prevented the metastatic bone lesions by inhibiting the mitogenic response of osteoblasts to growth factors produced by MDA-PCa 2b cells.

Activity of various hydroxylated vitamin D3 analogs for improving phosphorus utilisation in chicks receiving diets adequate in vitamin D3.

1. Young chicks were used to evaluate the efficacy of 2 new vitamin D3 analogs relative to 1 alpha-hydroxycholecalciferol (1 alpha-OH D3) for their ability to improve the bioavailability of phytate-bound phosphorus (P) when added to P-deficient maize-soyabean meal diets that were superadequate in cholecalciferol. 2. Both 20-epi-19-nor-1,25-(OH)2 D3 and 20-epi-19-nor-1 alpha-OH D3 were observed to have phytate-P releasing activity, as measured by bone ash responses. 3. However, the bioactivity of the 2 new analogs differed when compared with 1 alpha-OH D3. The 20-epi-19-nor-1 alpha-OH D3 analog had a lower (P < 0.05) phytate-P releasing activity (45%) than either 1 alpha-OH D3 of 20-epi-19-nor-1,25-(OH)2 D3, which did not differ. 4. A dietary concentration of 10 to 15 micrograms/kg 1 alpha-OH D3 was found to optimize phytate-P utilisation in 2-week-old chicks.

1alpha,25-dihydroxyvitamin D3-24-hydroxylase (CYP24) hydroxylates the carbon at the end of the side chain (C-26) of the C-24-fluorinated analog of 1alpha,25-dihydroxyvitamin D3.

The sequential oxidation and cleavage of the side chain of 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) initiated by the hydroxylation at C-24 is considered to be the major pathway of this hormone in the target cell metabolism. In this study, we examined renal metabolism of a synthetic analog of 1alpha,25(OH)2D3, 24, 24-difluoro-1alpha,25-dihydroxyvitamin D3 (F2-1alpha,25(OH)2D3), C-24 of which was designed to resist metabolic hydroxylation. When kidney homogenates prepared from 1alpha,25(OH)2D3-supplemented rats were incubated with F2-1alpha,25(OH)2D3, it was mainly converted to a more polar metabolite. We isolated and unequivocally identified the metabolite as 24,24-difluoro-1alpha,25,26-trihydroxyvitamin D3 (F2-1alpha,25,26(OH)3D3) by ultraviolet absorption spectrometry, frit-fast atom bombardment liquid chromatography/mass spectroscopy analysis, and direct comparison with chemically synthesized F2-1alpha,25,26(OH)3D3. Metabolism of F2-1alpha,25(OH)2D3 into F2-1alpha,25,26(OH)3D3 by kidney homogenates was induced by the prior administration of 1alpha,25(OH)2D3 into rats. The C-24 oxidation of 1alpha,25(OH)2D3 in renal homogenates was inhibited by F2-1alpha,25(OH)2D3 in a concentration-dependent manner. Moreover, F2-1alpha,25,26(OH)3D3 was formed in ROS17/2.8 cells transfected with a plasmid expressing 1alpha,25(OH)2D3-24-hydroxylase (CYP24) but not in the cells transfected with that expressing vitamin D3-25-hydroxylase (CYP27) or containing inverted CYP27 cDNA. These results show that CYP24 catalyzes not only hydroxylation at C-24 and C-23 of 1alpha,25(OH)2D3 but also at C-26 of F2-1alpha,25(OH)2D3, indicating that this enzyme has a broader substrate specificity of the hydroxylation sites than previously considered.

The action of various vitamin D3 metabolites on calcium and phosphorus metabolism in chick embryo calvariae.

Chick embryos from vitamin D-deficient hens given physiological doses of 1,25-dihydroxyvitamin D3 or 24,25-dihydroxyvitamin D3 or both become severely hypocalcemic, hyperphosphatemic and fail to hatch as compared to those derived from hens given 25-hydroxyvitamin D3 or 24,25-difluoro-25-hydroxyvitamin D3. Calvariae from the former contain less mineral and on incubation in vitro produce significantly lower calcium and higher phosphate concentration in the medium than do the calvariae derived from the embryos of hens supported on 25-hydroxyvitamin D3 or 24,24-difluoro-25-hydroxyvitamin D3.

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.

Effect of vitamin D3 metabolites on calcium and phosphorus metabolism in chick embryos.

The biochemical nature of the physiological defect found in chick embryos from hens supported on 1,25-dihydroxyvitamin D3 as their sole source of vitamin D is described. Vitamin D-deficient hens (44-wk-old) were divided into six groups of five and dosed daily for 19 wk with either 2.0 micrograms of 25-hydroxyvitamin D3, 2.0 micrograms of 24,24-difluoro-25-hydroxy-vitamin D3, 0.4 micrograms of 1,25-dihydroxyvitamin D3, 2.0 micrograms of 24,25-dihydroxyvitamin D3, 0.4 micrograms of 1,25-dihydroxyvitamin D3 plus 2.0 micrograms of 24,25-dihydroxyvitamin D3, or vehicle only. Normal embryonic development was found in eggs from hens given 25-hydroxyvitamin D3 or 24,24-difluoro-25-hydroxyvitamin D3, whereas embryos from hens given 1,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin D3, or their combination were abnormal and failed to hatch. Embryos from hens fed 1,25-dihydroxyvitamin D3 and/or 24,25-dihydroxyvitamin D3 had vitamin D deficiency: low bone ash, low plasma calcium, low total body calcium, and extremely high plasma phosphorus. Because the shell is the major source of calcium for the developing embryo, calcium transport from the shell to the embryos across the chorioallantoic membrane apparently fails, giving rise to the observed defects in embryonic development.

Stereo-retained and stereo-selective lactonization of four diastereoisomers of 23,25,26-trihydroxyvitamin D3 in homogenates of kidney from vitamin D-supplemented chicks.

To elucidate the biosynthesis of 25-hydroxyvitamin D3-26,23-lactone, various vitamin D3 derivatives were incubated individually with kidney homogenates prepared from vitamin D3-supplemented chicks, a preparation known to produce the 25-hydroxyvitamin D3-26,23-lactone from 25-hydroxyvitamin D3. The 25-hydroxyvitamin D3-26, 23-lactone produced in vitro was then separated, purified, identified, and quantitated by consecutive analysis by high-pressure liquid chromatography. The naturally occurring 23(S), 25(R)-25-hydroxyvitamin D3-26,23-lactone was produced from 23(S),25-dihydroxyvitamin D3, 25(R),26-dihydroxyvitamin D3, and 23(S),25(R),26-trihydroxyvitamin D3. 23(S),25 (S)-25-Hydroxyvitamin D3-26,23-lactone was synthesized from 25(S),26-dihydroxyvitamin D3 and 23(S),25(S),26-trihydroxyvitamin D3. The relative amounts of 25-hydroxyvitamin D3-26,23-lactones generated from the following vitamin D3 derivatives used as substrate (23(S),25(S),26-trihydroxyvitamin D3; 23(R),25(R),26-trihydroxyvitamin D3; 23(S),25(R),26-trihydroxyvitamin D3; 23(R),25(S),26-trihydroxyvitamin D3; 23(S), 25-dihydroxyvitamin D3; 23(R),25-dihydroxyvitamin D3; 25(S),26-dihydroxyvitamin D3; and 25(R),26-dihydroxyvitamin D3) are, respectively, 15:1.7:24:3.3:2.5:0:1:1.7. These results indicate that when the lactonization at C-23 and C-26 positions of various vitamin D3 derivatives occurred the stereochemical configuration at their C-23 and/or C-25 positions was not changed and the difference of the stereochemical configurations determined the rate of lactonization.

Isolation, identification, and metabolism of (23S,25R)-25-hydroxyvitamin D3 26,23-lactol. A biosynthetic precursor of (23S,25R)-25-hydroxyvitamin D3 26,23-lactone.

23S,25R,26-Trihydroxyvitamin D3 and (23S,25R)-25-hydroxyvitamin D3 26,23-lactol were chemically synthesized, and the metabolism of the two compounds to (23S,25R)-25-hydroxyvitamin D3 26,23-lactone in chick kidney homogenates was studied. 23S,25R,26-trihydroxyvitamin D3 was efficiently metabolized to the lactone in kidney homogenates from vitamin D-supplemented chicks, but not from vitamin D-deficient chicks. In contrast, the (23S,25R)-25-hydroxyvitamin D3 26,23-lactol was converted to the lactone in kidney homogenates regardless of the vitamin D status of the animals used. A new metabolite was isolated in pure form from the incubation mixture of 23S,25R,26-trihydroxyvitamin D3 with kidney homogenates prepared from vitamin D-supplemented chicks. The metabolite was identified as (23S,25R)-25-hydroxyvitamin D3 26,23-lactol by its ultraviolet and mass spectra and by derivatization. The structure was confirmed by direct comparison with an authentic sample on high pressure liquid chromatography. The evidence suggests that the stereochemistries of the isolated lactol at the 23- and 25-positions are S and R, respectively.

Biological activity of 24,24-difluoro-25-hydroxycholecalciferol in chicks.

The biological activity of subcutaneously injected 24,24-difluoro-25-hydroxycholecalciferol was compared with that of 25-hydroxycholecalciferol in the vitamin D-deficient growing chick. 24,24-Difluoro-25-hydroxycholecalciferol is equal to 25-hydroxycholecalciferol in the stimulation of: 1) growth, 2) intestinal calcium absorption, 3) elevation of serum calcium and serum phosphorus, 4) healing of rachitic cartilage (radiography), and 5) mineralization of rachitic bone (bone ash). The response appears to be linear in the range of 13.0 to 325 pmol daily. Since 24,24-difluorocholecaliferol cannot be 24-hydroxylated to produce either 24,25-dihydroxycholecalciferol or 1,24,25-trihydroxycholecalciferol, while it can be 1 alpha-hydroxylated to produce 24,24-difluoro-1,25-dihydroxycholecalciferol, these results demonstrate that 24-hydroxylation is not required for the known functions of cholecalciferol in the chick.

Isolation and identification of 1 alpha- and 23-hydroxylated metabolites of 25-hydroxy-24-oxovitamin D3 from in vitro incubates of chick kidney homogenates.

Five major metabolites (peaks I-V) of 25-hydroxy-24-oxovitamin D3 (25(OH)24-oxo-D3) have been isolated in pure form from in vitro incubates containing kidney homogenates of vitamin D-deficient chicks and chicks given 65 nmol of vitamin D3; peaks II, III, and V are from vitamin D-deficient chicks and peaks I, II, and IV are from vitamin D-supplemented birds. The structures of the metabolites were unequivocally identified as 23,25-dihydroxy-24-oxo-vitamin D3 (peak I), 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) (peak II), 1 alpha, 25-dihydroxy-24-oxovitamin D3 (peak III), 23,24,25-trihydroxyvitamin D3 (peak IV), and 1 alpha,24,25-trihydroxyvitamin D3 (peak V) by means of ultraviolet absorption spectrometry, mass spectrometry, and specific chemical reactions. It is concluded that 25(OH)24-oxo-D3 is further hydroxylated at the 1 alpha-position in the kidney of vitamin D-deficient chicks and at the 23-position in that of vitamin D-supplemented animals. Formation of 24,25(OH)2D3 from 25(OH)24-oxo-D3 in both vitamin D-deficient and -supplemented animals provides evidence for the presence of an enzyme to reduce the 24-oxo group irrespective of the vitamin D status.

Vitamin D3 toxicity in dairy cows.

Large parenteral doses of vitamin D3 (15 to 17.5 x 10(6) IU vitamin D3) were associated with prolonged hypercalcemia, hyperphosphatemia, and large increases of vitamin D3 and its metabolites in the blood plasma of nonlactating nonpregnant and pregnant Jersey cows. Calcium concentrations 1 day postpartum were higher in cows treated with vitamin D3 about 32 days prepartum (8.8 mg/100 ml) than in control cows (5.5 mg/100 ml). None of the cows treated with vitamin D3 showed signs of milk fever during the peripartal period; however, 22% of the control cows developed clinical signs of milk fever during this period. Signs of vitamin D3 toxicity were not observed in nonlactating nonpregnant cows; however, pregnant cows commonly developed severe signs of vitamin D3 toxicity and 10 of 17 cows died. There was widespread metastatic calcification in the cows that died. Because of the extreme toxicity of vitamin D3 in pregnant Jersey cows and the low margin of safety between doses of vitamin D3 that prevent milk fever and doses that induce milk fever, we concluded that vitamin D3 cannot be used practically to prevent milk fever when injected several weeks prepartum.

Chemical synthesis, biological activity, and metabolism of 25-hydroxy-24-oxovitamin D3.

25-Hydroxy-24-oxovitamin D3 (25(OH)24-oxo-D3), a metabolite of 25-hydroxyvitamin D3, has been chemically synthesized. The ultraviolet, mass, infrared, and proton nuclear magnetic resonance spectra of the 25(OH)24-oxo-D3 were identical with those of the natural product isolated from chick kidney incubates. The oxo compound showed biological activity similar to 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) in vitamin D-deficient chicks in enhancing intestinal calcium transport and bone calcium mobilization activities. Although 25(OH)24-oxo-D3 partially restored the impaired eggshell weights of Japanese quails fed a vitamin D-deficient diet, it was much less potent than 25-hydroxyvitamin D3 or 1 alpha, 25-dihydroxyvitamin D3. In addition, there was no effect on the calcification of medullary bone. When 25(OH)24-oxo[3H]D3 was incubated with kidney homogenates from vitamin D-deficient chicks, it was metabolized to [3H]1 alpha, 24,25-trihydroxyvitamin D3 and a metabolite which was eluted in a region between authentic 24R,25(OH)2D3 and 1 alpha, 25-dihydroxyvitamin D3 on high pressure liquid chromatography. In the incubates of kidney homogenates from vitamin D-supplemented chicks, those metabolites were not detected. In vitamin D-supplemented chicks, the recovery of radioactivity in the chloroform phase extracted by the method of Bligh and Dyer was only 50%, while that in vitamin D-deficient chicks was 87%. Moreover, the radioactivity eluted in the 25(OH)24-oxo-D3 fraction from vitamin D-supplemented chicks was only one-fifth of that from vitamin D-deficient birds. The present results indicate that the 24-oxidation of 24,25(OH)2D3 may be a route of inactivation of vitamin D3.