Synthesis of two carboxylic haptens for raising antibodies to 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3

Hapten derivatives of 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ were synthesized using the Wittig–Horner approach. Both haptens bearing a carboxylic group at the side chain that can be linked to a protein for raising antibodies of potential utility for the determination of 25-hydroxyvitamin D₃, 1α,25-dihydroxyvitamin D₃ and 1α-hydroxylated vitamin D₃ analogues.     

Tandem immunoaffinity chromatography for plasma 1α,25-dihydroxyvitamin D3 utilizing two antibodies having different specificities: A novel and powerful pretreatment tool for 1α,25-dihydroxyvitamin D3 radioreceptor assays

We report here a novel and powerful pretreatment method for radioreceptor assays (RRAs) for human plasma 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) based on “Tandem” immunoaffinity chromatography (Tandem IAC). Two antibodies having different specificities were each immobilized on agarose gel with cyanogen bromide to produce immunosorbents which were stable and repeatedly usable. An ethyl ether extract of plasma was applied to the first affinity column, from which 1,25(OH)₂D₃ could be preferentially eluted and separated from 1α-deoxy type metabolites. The effluent was then submitted to the second column, and the 1,25(OH)₂D₃ retained was eluted after non- or weakly-adsorbed interfering substances were washed out. This procedure allowed efficient purification without careful handling or strict time-management in the entire operation and enabled avoiding preparative high-performance liquid chromatography (HPLC) from RRA even with a conventional chick intestinal vitamin D receptor. Mean (± SD) plasma 1,25(OH)₂D₃ values of 56 normal subjects and 10 patients with chronic renal failure, obtained with this Tandem IAC/RRA system, were 36.4 (8.7) and 11.2 (4.0) pg/ml, respectively. The Tandem IAC will also be useful for developing immunoassays or gas chromatography-mass spectrometry of 1,25(OH)₂D₃.     

24R,25-dihydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3 are both indispensable for calcium and phosphorus homeostasis

The essential role of vitamin D throughout the life of most mammals and birds as a mediator of calcium homeostasis is well established. In view of the complex endocrine system existent for the regulated metabolism of vitamin D₃ to both 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] and 24R,25-dihydroxyvitamin D₃ [24R,25-(OH)₂D₃] (both produced by the kidney), an intriguing problem is to elucidate whether only one or both of these dihydroxyvitamin D₃ metabolites is required for the generation of all the biological responses mediated by the parent vitamin D₃. In contrast to the accumulated knowledge concerning the short term actions of 1,25(OH)₂-D₃ on stimulating intestinal calcium absorption and bone calcium reabsorption, relatively little is known of the biological function of 24,25(OH)₂D₃. We report now the results of a nine month study in which chicks were raised on a vitamin D-deficient diet from hatching to sexual maturity and received as their sole source of “vitamin D” either 24,25(OH)₂D₃ or 1,25(OH)₂D₃ singly or in combination. Specifically we are describing the integrated operation of the vitamin D endocrine system as quantitated by the individual measurement in all birds of 22 variables related to “vitamin D status” and as evaluated by the statistical procedure of multivariate discriminant analysis. Twelve of these variables involved detailed analysis of the bone including quantitative histology and the other 10 variables reflect various manifestations of vitamin D action, e.g. serum Ca²⁺ and Pi levels, vitamin D-dependent calcium binding protein (CaBP) in the intestine and kidney, egg productivity etc. As evaluated by the multivariate analysis, it is clear that 24,25(OH)₂D₃ and 1,25(OH)₂D₃ are simultaneously required for normalization of calcium homeostasis.     

Direct modulation of 25-hydroxyvitamin D3 hydroxylation in kidney tubules by 1,25-dihydroxyvitamin D3

Kidney tubules obtained from chicks fed a high-calcium low-phosphorus diet retained 25-hydroxyvitamin D₃-1-hydroxylase activity after a 10 h incubation in serum-free minimum essential medium. Inclusion of 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) in the medium prompted a suppression of 25-hydroxyvitamin D₃-1-hydroxylase and the induction of 25-hydroxyvitamin D₃-24-hydroxylase activities. The enzyme switch-over response could be prompted by 1.6 × 10^?7 M 1,25-dihydroxyvitamin D₃ and occurred within 6 h following treatment. Medium calcium appeared to augment the metabolite's switch-over action.     

Peritonitis induces the synthesis of 1α,25-dihydroxyvitamin D3 in macrophages from CAPD patients

Metabolism of 25-[3H]hydroxyvitamin D3 was studied in peritoneal macrophages from renal failure patients on continuous ambulatory peritoneal dialysis (CAPD). Cells from 5 out of 8 patients with a history of peritonitis produced significant amounts of a metabolite chromatographically identical to 1 alpha,25(OH)2D3; but none was produced by cells from non-infected patients. The evidence strongly suggests that peritoneal macrophages stimulated by infection can metabolise 25OHD3 to the active vitamin D3 metabolite, 1 alpha,25(OH)2D3, when maintained in short-term primary culture.     

Selective analogs of 1α,25-dihydroxyvitamin D3 for the study of specific functions of Vitamin D

New analogs of 1α,25-dihydroxyvitamin D₃ synthesized in our research group that show selective activity in vivo are presented along with supporting biological results. Compounds that act preferentially on intestine are 2-(3′-propylidene-19-nor-(20S or 20R))-1α,25-dihydroxyvitamin D₃ and 2-methylene-19-21-dinor-1α,25-dihydroxyvitamin D₃. Compounds that act anabolically to induce bone formation are 2-methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D₃ (2MD), the 2α-methyl derivative, the 26,27-dimethyl derivative, and the 26-dimethylene derivative. Compounds that act preferentially on parathyroid glands are 2-methylene-19-nor-1α-hydroxy-homopregnacalciferol, the 20S-bishomo derivative and the 2-methylene-19,26,27-trinor-1α,25-dihydroxyvitamin D₃. These latter compounds do not elevate serum calcium until doses of the order of >300 μg/kg body weight are used, while parathyroid hormone levels are suppressed at much lower doses. Some of these novel analogs may ultimately be useful as new and safer therapeutic agents. Regardless of their clinical utility, they represent valuable research tools that can be used to study the specific functions of the Vitamin D hormone in vivo.     

Differentiation induction of human leukemia cells (HL60) by a combination of 1,25-dihydroxyvitamin D3 and retinoic acid (all trans or 9-cis)

1,25(OH)₂D₃ and two stereoisomers of retinoic acid, all trans and 9-cis retinoic acid, are regulators of cell proliferation and differentiation. The aim of this study was to evaluate the effects of a combination of 1,25(OH)₂D₃ and retinoic acid (all trans or 9-cis) on proliferation and cell differentiation of the human promyelocytic leukemia cell line HL60, and to test the reversibility of the induced differentiation. Cell proliferation was inhibited as expected by 1,25(OH)₂D₃ and all trans retinoic acid alone (IC₅₀ of cell survival was 4 × 10⁻⁷ M, 9 × 10⁻⁶ M and 9 × 10⁻⁷ M for 1,25(OH)₂D₃, all trans and 9-cis retinoic acid, respectively). Combination of 1,25(OH)₂D₃ and either form of retinoic acid resulted in a partially additive decrease in cell proliferation. 1,25(OH)₂D₃ induced a monocytic differentiation (100% CD14+ cells with 10⁻⁷ M 1,25(OH)₂D₃), while retinoic acid led to a predominantly granulocytic differentiation (36 and 42% CD67+ cells with 10⁻⁶ M all trans and 9-cis retinoic acid, respectively). Additive effects on differentiation were observed upon combination of subtherapeutical doses of the drugs, achieving a mainly monocytic population, demonstrating the dominant role of 1,25(OH)₂D₃ in determining the direction of differentiation. The effects on proliferation and differentiation of the solitary drugs were reversible, while the proliferation arrest and differentiation induced by the combination persisted and even progressed after withdrawal of the drugs. We conclude that 1,25(OH)₂D₃ and retinoic acid (all trans or 9-cis) exert additive effects on inhibition of proliferation and induction of cell differentiation of HL60 cells, leading to a persistent differentiation, even after drug withdrawal.     

Evaluation of C-2-substituted 19-nor-1α,25-dihydroxyvitamin D3 analogs as therapeutic agents for prostate cancer

1α,25-Dihydroxyvitamin D₃ (1α,25(OH)₂D₃) is known to inhibit the proliferation and invasiveness of prostate cancer cells. However, 1α,25(OH)₂D₃can cause hypercalcemia and is not suitable as a therapeutic agent. 19-Nor-vitamin D derivatives are known to be less calcemic when administered systemically. In order to develop more potent anti-cancer agents with less calcemic side effect, we therefore utilized ³H-thymidine incorporation as an index for cell proliferation and examined the antiproliferative activities of nine C-2-substituted 19-nor-1α,25(OH)₂D₃ analogs in the immortalized PZ-HPV-7 normal prostate cell line. Among the nine analogs we observed that the substitution with 2α- or 2β-hydroxypropyl group produced two analogs having antiproliferative potency that is approximately 500- to 1000-fold higher than 1α,25(OH)₂D₃. The ³H-thymidine incorporation data were supported by the cell counting data after cells were treated with 1α,25(OH)₂D₃, 19-nor-2α-(3-hydroxypropyl)-1α,25(OH)₂D₃ or 19-nor-2β-(3-hydroxypropyl)-1α,25(OH)₂D₃ for 7 days. 19-Nor-2α-(3-hydroxypropyl)-1α,25(OH)₂D₃ and 19-nor-2β-(3-hydroxypropyl)-1α,25(OH)₂D₃ were also shown to be about 10-fold more active than 1α,25(OH)₂D₃ in cell invasion studies using prostate cancer cells. In conclusion, a substitution at the C-2 position of 19-nor-1α,25(OH)₂D₃ molecule with a hydroxypropyl group greatly increased the antiproliferative and anti-invasion potencies. Thus, these two analogs could be developed to be effective therapeutic agents for treating early and late stages of prostate cancer.     

Ring A-stereoisomers of 1-hydroxyvitamin D3 and their relative binding affinities for the intestinal 1α,25-dihydroxyvitamin D3 receptor protein

A set of eight 1-hydroxyvitamin D₃ compounds comprising the four possible (5Z)-1,3-diol stereoisomers and the corresponding (5E)-double bond isomers, has been prepared in order to assess the effect of 1,3-diol stereochemistry and 5,6-double bond geometry on binding affinity for the intestinal 1,25-(OH)₂D₃-receptor protein. The compounds were synthesized from either vitamin D₃ or 3-epivitamin D₃ via 3,5-cyclovitamin D intermediates. Competitive receptor binding assays establish that all changes from the natural ring A-configuration (1S, 3R, 5Z) lead to decreased binding affinity, and confirm the importance of the 1-hydroxy function since the conversion of stereochemistry at that center from 1α(S) to 1β(R) has the most pronounced effect on binding affinity (attenuation by more than three orders of magnitude). Other modifications (i.e., conversion at C-3, or cis to trans isomerization of the 5,6-double bond) decrease binding affinity by more moderate (ca. 10-fold) but cumulative factors.     

Extra-renal 25-hydroxyvitamin D3-1α-hydroxylase in human health and disease

Although ectopic expression of 25-hydroxyvitamin D₃-1α-hydroxylase (1α-OHase) has been recognized for many years, the precise function of this enzyme outside the kidney remains open to debate. Three specific aspects of extra-renal 1α-OHase have attracted most attention: (i) expression and regulation in non-classical tissues during normal physiology; (ii) effects on the immune system and inflammatory disease; (iii) expression and function in tumors. The most well-recognized manifestation of extra-renal 1α-OHase activity remains that found in some patients with granulomatous diseases where locally synthesized 1α,25(OH)₂D₃ has the potential to spill-over into the general circulation. However, immunohistochemistry and mRNA analyses suggest that 1α-OHase is also expressed by a variety of normal human tissues including the gastrointestinal tract, skin, vasculature and placenta. This has promoted the idea that autocrine/paracrine synthesis of 1,25(OH)₂D₃ contributes to normal physiology, particularly in mediating the potent effects of vitamin D on innate (macrophage) and acquired (dendritic cell) immunity. We have assessed the capacity for synthesis of 1,25(OH)₂D₃ in these cells and the functional significance of autocrine responses to 1α-hydroxylase. Data suggest that local synthesis of 1,25(OH)₂D₃ may be a preferred mode of response to antigenic challenge in many tissues.     

Determination of prostaglandin F2α, E2, D2 and 6-keto-F1α in human cerebrospinal fluid

Prostaglandin (PG)F_2α, E₂, D₂ and 6-keto-F_1α were determined in human cerebrospinal fluid by a mass spectrometric technique. The samples were obtained from 12 patients with suspected intracranial disease. A 64 fold variation in PG levels was observed. The major PG was 6-keto-F_1α (0.12–15 ng/ml). PGF_2α and PGE₂ were present in lower concentrations PGD₂ was below the level of detection (0.05 ng/ml) except in one patient with extremely high total levels of PGs.     

Simplified method for the determination of 25-hydroxy and 1α,25-dihydroxy metabolites of vitamins D2 and D3 in human plasma application to nutritional studies

A simplified method for the determination of 25-hydroxy and 1α,25-dihydroxy metabolites of vitamins D₂ and D₃ in human plasma was developed. Plasma samples were deproteinizated and applied to a Bond Elut C₁₈ OH cartridge to separate 25-hydroxyvitamin D (25-OH-D) and 1α-25-dihydroxyvitamin D [1,25(OH)₂D] fractions. The 25-OH-D fraction was purified by a Bond Elut C₁₈ cartridge and 25-OH-D₂ and 25-OH-D₃ were assayed by HPLC using a Zorbax SIL column. The 1,25(OH)₂D fraction obtained above was subsequently applied to HPLC using a Zorbax SIL column to separate 1,25(OH)₂D₂ and 1,25(OH)₂D₃ fractions which were determined by a radioreceptor assay (RRA) using calf thymus receptor. The method was applied to nutritional studies.     

1α,25-dihydroxy-24-oxo-16-ene vitamin D3, a metabolite of a synthetic vitamin D3 analog, 1α,25-dihydroxy-16-ene vitamin D3, is equipotent to its parent in modulating growth and differentiation of human leukemic cells

1α,25(OH)₂-16-ene-D₃, a synthetic analog of the steroid hormone, 1α,25(OH)₂D₃, has great potential to become a drug in the treatment of leukemia and other proliferative disorders, because of its minimal in vivo calcemic activity associated with a potent inhibitory effect on cell growth. However, at present, the mechanisms through which 1α,25(OH)₂-16-ene-D₃ expresses its biological activities are still not completely understood. Our previous in vitro study in a perfused rat kidney indicated for the first time that 1α,25(OH)₂-16-ene-D₃ and 1α,25(OH)₂D₃ are metabolized differently. 1α,25(OH)₂-24-oxo-16-ene-D₃, an intermediary metabolite of 1α,25(OH)₂-16-ene-D₃ formed through the C-24 oxidation pathway, accumulated significantly in the perfusate when compared to 1α,25(OH)₂-24-oxo-D₃, the corresponding intermediary metabolite of 1α,25(OH)₂D₃. In a subsequent in vivo study, we also reported that 1α,25(OH)₂-24-oxo-16-ene-D₃ exerted immunosuppressive activity equal to its parent, without causing significant hypercalcemia. In order to establish further the critical role of 1α,25(OH)₂-24-oxo-16-ene-D₃, in generating some of the key biological activities ascribed to its parent, we performed the present in vitro study using a human myeloid leukemic cell line (RWLeu-4) as a model. Comparative target tissue metabolism studies indicated that 1α,25(OH)₂-16-ene-D₃ and 1α,25(OH)₂D₃ are metabolized differently in RWLeu-4 cells, and the differences were similar to the ones we previously observed in the rat kidney. The significant finding was the accumulation of 1α,25(OH)₂-24-oxo-16-ene-D₃ in RWLeu-4 cells because of its resistance to further metabolism. Biological activity studies indicated that both 1α,25(OH)₂-16-ene-D₃ and its 24-oxo metabolite produced growth inhibition and promoted differentiation of RWLeu-4 cells to the same extent, and these activities were several fold higher than those exerted by 1α,25(OH)₂D₃. In addition, the genomic action of each vitamin D compound was assessed in a rat osteosarcoma cell line (ROS 17/2.8) by measuring its ability to transactivate a gene construct containing the vitamin D response element of the osteocalcin gene linked to the growth hormone reporter gene. In these studies, both 1α,25(OH)₂-16-ene-D₃ and its 24-oxo metabolite exerted similar but potent transactivation activity which was several fold greater than that exerted by 1α,25(OH)₂D₃ itself. In summary, our results indicate that the production and slow clearance of the bioactive intermediary metabolite, 1α,25(OH)₂-24-oxo-16-ene-D₃, in RWLeu-4 cells contributes significantly to the final expression of the enhanced biological activities ascribed to its parent analog, 1α,25(OH)₂-16-ene-D₃.     

1α,25-dihydroxyvitamin D3 hybrid analogs with structural changes at both the A-ring and the C,D-ring side-chain

Calcitrol analogs 5, designed to combine two remote structural changes each of which separately produces a sharply different biological profile, have biological activities that are a blending of the effects of each structural change.     

1α,25(OH)2-vitamin D3 membrane-initiated calcium signaling modulates exocytosis and cell survival

1α,25(OH)₂-vitamin D₃ (1,25D) is considered a bone anabolic hormone. 1,25D actions leading to bone formation involve gene transactivation, on one hand, and modulation of cytoplasmic signaling, on the other. In both cases, a functional vitamin D receptor (VDR) appears to be required. Here we study 1,25D-stimulated calcium signaling that initiates at the cell membrane and leads to exocytosis of bone materials and increased osteoblast survival. We found that rapid 1,25D-induction of exocytosis couples to cytoplasmic calcium increase in osteoblastic ROS 17/2.8 cells. In addition, we found that elevation of cytoplasmic calcium concentration is involved in 1,25D anti-apoptotic effects via Akt activation in ROS 17/2.8 cells and non-osteoblastic CV-1 cells. In both cases, 1,25D-stimulated elevation of intracellular calcium is due in part to activation of L-type Ca²⁺ channels. We conclude that 1,25D bone anabolic effects that involve increased intracellular Ca²⁺ concentration in osteoblasts can be explained at two levels. At the single-cell level, 1,25D promotes Ca²⁺-dependent exocytotic activities. At the tissue level, 1,25D protects osteoblasts from apoptosis via a Ca²⁺-dependent Akt pathway. Our studies contribute to the understanding of the molecular basis of bone diseases characterized by decreased bone formation and mineralization.     

Biphasic action of prostaglandin E2 on conversion of 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 in chick renal tubules

The effect of PGE₂ on the conversion of 25-hydroxyvitamin D₃ (25 OH D₃) to 1,25-dihydroxyvitamin D₃ (1,25- (OH) ₂D₃) by isolated renal tubules from vitamin D deficient chicks was studied under a variety of experimental conditions. In the absence of added vitamin D metabolites, PGE₂ (2 × 10⁻⁶M) caused an immediate inhibition of formation of 1,25-(OH) ₂D₃, followed by a delayed stimulation, apparent after 15 h exposure to PGE₂. Pretreatment of the tubules with 1,25-(OH) ₂D₃ prevented the immediate inhibitory action of PGE₂, and allowed the stimulation to be apparent after 4 h exposure to PGE₂. The cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1-methyl xanthine (IBMX) significantly stimulated the formation of 1,25-(OH) ₂D₃. PGE₂ significantly inhibited 1,25-(OH) ₂D₃ formation in tubules which had been stimulated by IBMX. PGE₂ stimulated the adenylate cyclase activity in a crude particulate fraction from the chick kidney, and raised cyclic adenosine 3′, 5′-monophosphate (cyclic AMP) levels in the renal tubules.It is concluded that PGE₂ can either stimulate or inhibit 1,25-(OH) ₂D₃ formation in chick renal tubules. The stimulatory effect may be partly due to elevation of cyclic AMP. The mechanism of the inhibitory effect requires further investigation.     

The clearance of human fibrinogen fragments D1, D2, D3 and fibrin fragment D1 dimer in mice

The clearance of human fibrinogen fragments D₁, D₂, D₃ and fibrin fragment D₁ dimer were studied in the mouse model. Clearance of these fragments is a complex process involving clearance from blood into three other compartments. The overall clearance of fragment D₁ and its dimer were essentially identical. Fragments D₂ and D₃ cleared at a progressively slower rate. Competition studies were performed between ¹²⁵I-labeled fragment D₁ and large molar excesses of unlabelled human fragments D₁, D₂, D₃, D₁ dimer, fragment E, fibrinogen, macroalbumin, mannan and asialooroscomucoid. Of these ligands only the fragment D variants competed for the clearance of ¹²⁵I-labeled fragment D₁. Cross-competition was observed when ¹²⁵I-labeled fragment D₁ dimer was cleared in the presence of large molar excesses of fragment D₁. Autopsies demonstrated that injected fragments D₁, D₂, D₃ and D₁ dimer cleared primarily in liver and kidneys. In some clearance studies, livers were perfused with tissue culture fluid, subjected to light microscopic autoradiography, and silver grain counts performed to localize cleared fragment D₁. These experiments indicated that 80% of the liver uptake was in hepatocytes. However, when silver grain counts were normalized for the number of parenchymal and nonparenchymal cells, the distribution of silver grains was essentially identical (1.8 and 1.6 grains per cell, respectively). It is concluded that fragments D₁, D₂, D₃ and D₁ dimer are recognized by a similar clearance pathway. Since neither fibrinogen nor fragment E competed for the clearance of fragment D₁, it is suggested that determinants present in the fragment D domain become exposed after plasmin attack on fibrinogen and are responsible for clearance.