Potent Vitamin Research Articles

An improved synthesis of 24,24-difluoro-1.ALPHA.,25-dihydroxyvitamin D3 from vitamin D2.

An improved synthesis of a highly potent vitamin D3 analog, 24, 24-difluoro-1α, 25-dihydroxyvitamin D3 has been accomplished. The total yield was 9.3% from inexpensive vitamin D2 in 11 steps.

Nonhypercalcemic 1,25-(OH)2D3 analogs potently induce the human osteocalcin gene promoter stably transfected into rat osteosarcoma cells (ROSCO-2)

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] is the active hormonal form of vitamin D3 and has potent effects on bone and calcium regulation. Over the past decade it has become apparent that 1,25-(OH)2D3 has other effects on cellular proliferation that potentially could be developed for therapy in human malignancy. Since the hypercalcemic effects of 1,25-(OH)2D3 have limited that use in the human, novel nonhypercalcemic analogs of 1,25-(OH)2D3 have been synthesized. The molecular mechanism of this divergence in these antiproliferative and calcium-regulating actions is unexplained. We have previously examined the human bone-specific gene osteocalcin as a model of the molecular mechanisms of vitamin D action in bone and have shown that induction of the osteocalcin gene by 1,25-(OH)2D3 is mediated through an unique and complex palindromic region of the promoter similar to but distinct from those of other steroid hormone-responsive elements. Using an osteosarcoma cell line permanently transfected with the vitamin D-responsive promoter of the human osteocalcin gene linked to a "reporter" gene, we have shown that there is a dose-dependent induction of CAT activity by 1,25-(OH)2D3 and that the potencies of vitamin D metabolites and analogs are comparable to those found in other vitamin D bioassays. Furthermore, vitamin D analogs, including MC-903, 22-oxa-1,25-(OH)2D3, and delta 22-1,25S,26-trihydroxyvitamin D3, which effect cellular differentiation but lack hypercalcemic activity in vivo, exhibit osteocalcin promoter inductive actions virtually identical to those of 1,25-(OH)2D3. Consideration of these and other data support the hypothesis that the divergent effects of such analogs on differentiation and calcium homeostasis reflect pharmacokinetic differences in vivo rather than distinct 1,25-(OH)2D3-sensitive pathways.

Protein S and C alterations in acutely III patients

Protein C, a potent vitamin K-dependent protein activated by an endothelial cell cofactor, thrombomodulin, has anticoagulant and profibrinolytic activity. Free protein S, a cofactor for protein C, potentiates protein C activity at the endothelial cell surface. Pulmonary thromboemboli are a consistent finding in adult respiratory distress syndrome (ARDS). To determine if protein S or protein C were affected by widespread endothelial cell damage in ARDS, we measured bound and free protein S levels and protein C antigenic and functional levels in 18 patients with acute lung injury, 6 critically ill patients without lung history, and 22 normal subjects. Free (PS:F) and bound (PS:Ag) protein S and protein C antigen (PC:Ag) levels were measured using an enzyme-linked immunoassay and protein C function (PC:Fn) by measuring its anticoagulant activity. We found a significant decrease in bound and free protein S levels of both patient groups in comparison to normal and a shift toward the inactive, bound protein S form. In addition, a significant decrease in free protein S compared to bound protein S in both patient groups was observed. While both PC:Ag and PC:Fn were significantly reduced compared to normal, the PC:Fn was significantly and severely decreased out of proportion to the PC:Ag in both patient groups. There was no difference between those with and without lung injury for both protein S and protein C. Analyzed according to etiology of lung injury, there was no difference in the bound and free protein S, nor in PC:Ag and PC:Fn levels between patients with sepsis and trauma. However, there were significant decreases in both protein S and protein C levels compared with normal subjects. Levels of both PS and PC levels in patients who survived did not differ from those who died. In summary, our data show that both protein S and C are markedly deranged in acutely ill patients who suffered from either sepsis or trauma, and these changes are independent of lung injury. The marked reductions in functional activity of PS and PC may be contributing factors to the thromboembolic complications often observed in these patients.

Surreptitious Ingestion of a Long-Acting Vitamin K Antagonist/Rodenticide, Brodifacoum: Clinical and Metabolic Studies of Three Cases

Abstract The vitamin K metabolism of three patients with factitious purpura due to brodifacoum ingestion was studied. These patients, who presented with bleeding disorders due to deficiency of the vitamin K-dependent blood clotting proteins, were refractory to vitamin K1 at standard doses and required fresh frozen plasma to control bleeding until large doses of vitamin K1 were used. Metabolic studies demonstrated a blockade in vitamin K utilization, consistent with the presence of a vitamin K antagonist, but the patients denied use of anticoagulants. Warfarin assays were negative. We show that the factitious purpura in each patient was due to the surreptitious ingestion of brodifacoum, a potent second generation long-acting vitamin K antagonist used as a rodenticide. The coagulopathies responded to long-term therapy with large doses of vitamin K1. The serum elimination half-time for brodifacoum ranged from 16 to 36 days in these patients. The anticoagulant effect is of long duration, requiring chronic vitamin K treatment. With increasing availability of new rodenticides, factitious purpura due to surreptitious ingestion of these potent vitamin K antagonists is emerging as a new problem, previously associated with warfarin, with important implications for diagnosis and treatment.

Surreptitious ingestion of a long-acting vitamin K antagonist/rodenticide, brodifacoum: clinical and metabolic studies of three cases

The vitamin K metabolism of three patients with factitious purpura due to brodifacoum ingestion was studied. These patients, who presented with bleeding disorders due to deficiency of the vitamin K-dependent blood clotting proteins, were refractory to vitamin K1 at standard doses and required fresh frozen plasma to control bleeding until large doses of vitamin K1 were used. Metabolic studies demonstrated a blockade in vitamin K utilization, consistent with the presence of a vitamin K antagonist, but the patients denied use of anticoagulants. Warfarin assays were negative. We show that the factitious purpura in each patient was due to the surreptitious ingestion of brodifacoum, a potent second generation long-acting vitamin K antagonist used as a rodenticide. The coagulopathies responded to long-term therapy with large doses of vitamin K1. The serum elimination half-time for brodifacoum ranged from 16 to 36 days in these patients. The anticoagulant effect is of long duration, requiring chronic vitamin K treatment. With increasing availability of new rodenticides, factitious purpura due to surreptitious ingestion of these potent vitamin K antagonists is emerging as a new problem, previously associated with warfarin, with important implications for diagnosis and treatment.

The synthesis and biological activity of 25-hydroxy-26,27-dimethylvitamin D 3 and 1,25-dihydroxy-26,27-dimethylvitamin D 3: highly potent novel analogs of vitamin D 3

We synthesized 25-hydroxy-26,27-dimethylvitamin D 3, 9, and 1,25-dihydroxy-26,27-dimethylvitamin D 3,14, from chol-5-enic acid-3β-ol and tested their biological activity in vivo and in vitro. 9 was found to be a highly potent vitamin D analog with bioactivity similar to that of 25-hydroxyvitamin D 3. 9 bound to rat plasma vitamin D binding protein with approximately one-third the affinity of 25-hydroxyvitamin D 3. In a duodenal organ culture system and in a competitive binding assay with chick intestinal 1,25-dihydroxyvitamin D receptor, 9 was significantly more potent than 25-hydroxyvitamin D 3. 1,25-Dihydroxy-26,27-dimethylvitamin D 3, 14 was also highly active in vivo. At doses of 1000–5000 pmol/rat, its action was more sustained than that of 1,25-dihydroxyvitamin D 3.14 bound to vitamin 0 binding protein about 18 times less effectively than 1,25-dihydroxyvitamin D 3. 14 bound to the chick intestinal cytosol receptor with an affinity one-half that of 1,25-dihydroxyvitamin D 3. In a duodenal organ culture system, 14 was about half as active as 1,25-dihydroxyvitamin D 3. Extension of the sterol side chain, at C-26 and C-27, by methylene groups, prolongs the bioactivity of a vitamin D sterol hydroxylated at C-1 and C-25; the corresponding sterol, hydroxylated only at C-25, does not show any alteration of its bioactivity in vivo. These newly synthesized analogs may potentially be of therapeutic use in various mineral disorders.

The synthesis and biological activity of 22-fluorovitamin D 3: A new vitamin D analog

We synthesized 22-fluorovitamin D 3 from (22S) cholest-5-ene-3β, 22-diol-3β-acetate 2 . Compound 2 was treated with diethylaminosulfur trifluoride to give 22-fluorocholest-5-en-3β-acetate 3 and (E) 22-dehydrocholest-5-en-3β-acetate. Compound 3 was treated with N-bromosuccinimide to give a mixture of the respective 5,7- and 4,6-dienes. The 5,7-diene of 3 was separated from the 4,6-diene using the dienophile 4-phenyl-1,2,4-triazoline-3,5-dione. 22-Fluoro-5α, 8α-(3,5-dioxo-4-phenyl-1,2,4-triazolino)-cholest-6-en-3β-acetate 4 was purified by flash chromatography and treated with lithium aluminum hydride to generate 22-fluorocholesta-5,7-dien-3β-ol 5 . Photolysis of the diene 5 , followed by thermal equilibration, resulted in the synthesis of 22-fluorovitamin D 3 7 . The vitamin 7 increased active intestinal calcium transport only at a dose of 50,000 pmol/rat, whereas vitamin D 3 increased intestinal calcium transport at a dose of between 50 and 500 pmol/rat. 22-Fluorovitamin D 3 7 did not mobilize bone and soft tissue calcium at a dose as high as 50,000 pmol/rat, whereas vitamin D 3 was successful in doing so at a dose of 500 pmol/rat. When tested in the duodenal organ culture system, 22-fluorovitamin D 3 7 had approximately 1/25th the potency of vitamin D 3. It did not antagonize the activity of 1,25-dihydroxyvitamin D 3. 22-Fluorovitamin D 3 7 bound to the rat plasma vitamin D binding protein less avidly than vitamin D 3. 22-Fluorovitamin D 3 was bound very poorly to the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D 3. We conclude that the introduction of fluorine at the C-22 position results in a vitamin D sterol with decreased biologic activity when compared to vitamin D 3. The presence of a fluorine group at C-22 position inhibits the binding of the vitamin to rat vitamin D binding protein when compared to the binding of its hydrogen analog, vitamin D 3.

Regulation by vitamin D metabolites of messenger ribonucleic acid for preproparathyroid hormone in isolated bovine parathyroid cells.

We have recently determined that high calcium concentrations, in parallel with their suppressive effects on parathyroid hormone (PTH) secretion, reversibly and specifically decrease preproPTH mRNA in cultured bovine parathyroid cells. In order to determine whether vitamin D metabolites also regulate the content of preproPTH mRNA, we tested their effects on bovine parathyroid cells in the same culture system. Levels of preproPTH mRNA were determined by dot-blot hybridization or blot hybridization with a labeled cloned cDNA probe. Incubation with 1,25-dihydroxycholecalciferol at doses varying from 10 pM to 0.1 microM caused a direct decrease in mRNA down to 50% of control values at 48 hr. There was no evidence that 1,25-dihydroxycholecalciferol, even at the highest concentrations, had any toxic effects on cell number or viability or on total RNA or RNA synthesis. Levels of alpha-actin mRNA did not change in the same experiments, and the suppression of preproPTH mRNA was reversible. When the relative potency of various vitamin D metabolites in suppressing preproPTH mRNA was evaluated, 1,25-dihydroxycholecalciferol greater than 24,25-dihydroxycholecalciferol greater than 25-hydroxycholecalciferol greater than vitamin D3 (cholecalciferol). These effects were highly specific and suggest that vitamin D metabolites play an important role in regulating the production of PTH.

Antineoplastic potency of vitamin C on benzo(a)pyrene-induced tumours : G. Kallistratos, E. Fasske, A. Donos and A. Evangelou. Department of Experimental Physiology, Faculty of Medicine, University of Ioan, Ioannina, Greece, and Department of Pathology, Research Institute of Experimental Biology and Medicine, D-2061 Borstel, F.R.G.

Antineoplastic potency of vitamin C on benzo(a)pyrene-induced tumours : G. Kallistratos, E. Fasske, A. Donos and A. Evangelou. Department of Experimental Physiology, Faculty of Medicine, University of Ioan, Ioannina, Greece, and Department of Pathology, Research Institute of Experimental Biology and Medicine, D-2061 Borstel, F.R.G.

Dark adaptation of patients with head and neck tumors

20 patients with tumors of the head and neck region have a delayed dark adaption. The serum vitamin A levels in these patients are significantly diminished versus controls. Since disturbance of dark adaption does not occur before long during deficiency of vitamin A, it is assumed that the deficiency was existent long time before. With regard to the possible antineoplastic potency of vitamin A, this is considered a possibly important factor in tumor development and growth. The disturbed vitamin A metabolism is possibly due to a long lasting imbalanced diet.

Interaction between 24R,25-dihydroxycholecalciferol and 1,25-dihydroxycholecalciferol on 45Ca release from bone in vitro.

Interaction among vitamin D3 metabolites on bone receptor sites is not known. Therefore, interaction between the most potent vitamin D3 metabolite, 1,25(OH)2D3, and the most abundant dihydroxymetabolite, 24R,25(OH)2D3, was studied on isolated rat fetal bone by measuring 45Ca release from prelabeled bones. 24R,25(OH)2D3 at concentrations of 10-50 ng/ml caused marked inhibition of the bone-resorbing activity of 1,25(OH)2D3 at concentrations of 10-50 pg/ml. 24S,25(OH)2 (unnatural enantiometer), on the other hand, at a concentration of 100 ng/ml did not inhibit the bone-resorbing effect of 10 pg/ml 1,25(OH)2D3. 24R,25(OH)2D3 at a concentration of 20 ng/ml did not inhibit the 45Ca-releasing effect of a submaximal concentration of PTH (500 ng/ml). Therefore, the inhibitory effect of 24R,25(OH)2D3 on the bone response to 1,25(OH)2D3 appeared to be specific and probably due to a competitive inhibitory effect. In addition, the inhibitory effect of 24R,25(OH)2D3 was weak, since it could be partially overcome by increasing the concentration of 1,25(OH)2D3.

Induction of calcium-binding protein in organ-cultured chick intestine by fluoro analogs of vitamin D 3

Vitamin D-like steroids added to the culture medium induce a specific calcium-binding protein (CaBP) in embryonic chick duodenum maintained in organ culture. This system provides a biologically relevant assay, i.e., a physiological response in a principle target organ, for the study of the relative biopotency of vitamin D metabolites and analogs. A number of fluoro analogs of vitamin D 3 (D 3) and its metabolites were assayed in the present study. Analogs fluorinated in the lα position (1α-F-D 3) or in both the 1α and 25 positions (1α,25-F 2-D 3) were markedly more potent than vitamin D 3 itself although 1α,25-F 2-D 3 was only 1 7 th as potent as 1α-F-D 3. The 25-fluoro analog (25-F-D 3) was a very weak inducer; only 1 45 th as potent as vitamin D 3. The 25-fluoro analog of 1α-hydroxyvitamin D 3 (1α-OH-25-F-D 3) was less potent than its nonfluorinated counterpart. Although 25-fluorination reduced biopotency in all other analogs tested, 24 R-OH-25-F-D 3 was about 15 times more potent than 24 R,25-(OH) 2-D 3. Of considerable interest was the effect of difluorination at the 24-carbon position: both 24,24-F 2-25-OH-D 3 and 24,24-F 2-1α,25-(OH) 2-D 3 were about four times as potent as their nonfluorinated counterparts. The 24,24-F 2-1α,25-(OH) 2-D 3 is, therefore, the most potent vitamin D 3 analog yet tested in this system i.e., it is four times more potent than the most potent naturally occurring vitamin D 3 metabolite, 1α,25-(OH) 2-D 3.

The influence of vitamin E quinone on platelet structure, function, and biochemistry

Although the effects of vitamin E on platelet function have been investigated in vivo and in vitro, vitamin E quinone, a natural metabolite of vitamin E, has been virtually overlooked. This oxidized form of vitamin E inhibits platelet aggregation and secretion induced by various aggregating agents more effectively than vitamin E by a magnitude of 5–10-fold. Vitamin E and vitamin E quinone do not alter platelet ultrastructure or cellular concentrations of serotonin and adenine nucleotides, including cAMP. Inhibition of aggregation by vitamin E quinone occurs in the absence of detectable reduction of vitamin E quinone or oxidation of vitamin E and is readily reversed by washing the platelet. Only vitamin E quinone prevents arachidonic acid release and slightly inhibits cyclooxygenase, whereas both agents partially prevent calcium release from a platelet subcellular organelle. Vitamin E quinone also inhibited synthesis of prostacyclin by endothelial cells with basal synthesis in the presence of external arachidonic acid being less affected than thrombin-stimulated PGI2 production. The greater potency of vitamin E quinone in suppressing platelet function compared to vitamin E suggests that this quinone metabolite may be the better antithrombotic agent and possibly responsible for in vivo effects previously attributed to vitamin E.

The Influence of Vitamin E Quinone on Platelet Structure, Function, and Biochemistry

Although the effects of vitamin E on platelet function have been investigated in vivo and in vitro, vitamin E quinone, a natural metabolite of vitamin E, has been virtually overlooked. This oxidized form of vitamin E inhibits platelet aggregation and secretion induced by various aggregating agents more effectively than vitamin E by a magnitude of 5-10-fold. Vitamin E and vitamin E quinone do not alter platelet ultrastructure or cellular concentrations of serotonin and adenine nucleotides, including cAMP. Inhibition of aggregation by vitamin E or vitamin E quinone occurs in the absence of detectable reduction of vitamin E quinone or oxidation of vitamin E and is readily reversed by washing the platelets. Only vitamin E quinone prevents arachidonic acid release and slightly inhibits cyclooxygenase, whereas both agents partially prevent calcium release from a platelet subcellular organelle. Vitamin E quinone also inhibited synthesis of prostacyclin by endothelial cells with basal synthesis in the presence of external arachidonic acid being less affected than thrombin-stimulated PGI2 production. The greater potency of vitamin E quinone in suppressing platelet function compared to vitamin E suggests that this quinone metabolite may be the better antithrombotic agent and possibly responsible for in vivo effects previously attributed to vitamin E.

The influence of substitution at C 24 on the calcium-binding protein-stimulating activity of vitamin D metabolites in chick embryonic duodenum

The production of calcium-binding protein, in vitro, by embryonic chick duodenum has been used to assess the potency of vitamin D compounds. The introduction of an hydroxyl on 1-, 25-, or 24R-position enhanced biological activity while the introduction of both 1α- and 25-hydroxyls produced maximal activity. However 24R-hydroxylation of 1,25-dihydroxyvitamin D 3 diminished activity. The vitamin D 2 side chain on 25-hydroxyvitamin D or 1,25-dihydroxyvitamin D did not greatly diminish activity in contrast to the fact that the vitamin D 2 compounds are 10% as active as the vitamin D 3 compounds in vivo in the chick. These results support the idea that the target organs of the chick do not discriminate against the vitamin D 2 side chain and that the discrimination in this species is at the level of metabolism.

Variable Potency of Intramuscular Vitamin D Preparations

Variable Potency of Intramuscular Vitamin D Preparations

1α‐hydroxyvitamin D3 Treatment of Therapy‐Resistant Symptomatic Hypocalcemia in a Hypoparathyroid Patient with Intestinal Malabsorption

The case history of a hypoparathyroid female with short bowel syndrome and long-standing therapy-resistant symptomatic hypocalcemia is reported. During treatment with massive doses of the potent vitamin D analog, 1 alpha-hydroxyvitamin D3(1 alpha(OH)D3), normocalcemia was re-established and clinical symptoms of hypocalcemia were relieved. Furthermore, significant improvement of t of intestinal calcium absorption and bone mineral content was observed after three months of treatment with 1 alpha(OH)D3. The data suggest that 1 alpha(OH)D3 may be of therapeutical value in patients with hypoparathyroidism and intestinal malabsorption.

The investigation and treatment of renal bone disease

Neutron activation analysis of a hand has been used to monitor changes in skeletal calcium content of patients undergoing maintenance hemodialysis. Assessed by this technic supplementary magnesium therapy was of value in reducing the progressive losses of skeletal calcium in a proportion of the patients receiving maintenance hemodialysis. Oral calcium supplements produced a decrease in the skeletal calcium losses of all the patients studied. 1-alpha-hydroxycholecalciferol, a potent vitamin D analogue, caused a significant increase in the calcium content of bone in three patients receiving maintenance hemodialysis; it may prove to be a safe and effective form of therapy for azotemic osteodystrophy.

Specificity of cellular retinol-binding protein for compounds with vitamin A activity.

THE importance of vitamin A (retinol) for normal growth and development of vertebrates is well established1 but its mechanism(s) of action, except in vision, remains obscure. Retinol can influence differentiation of epithelial cells2 and is required for normal development of the testis and for maintenance of pregnancy3, suggesting a regulatory role resembling that of certain hormones in some aspects of its activity. The action of many steroid hormones is apparently mediated through specific cellular binding proteins4. We have found that rat tissues5,6 and human5 and rabbit lungs7 contain a protein able to bind all-trans-retinol, the most potent vitamin A compound, with high affinity and specificity. In all species studied so far, this protein has the same apparent size (molecular weight 14,000, sedimenting in the 2S region on sucrose gradients) and seems to carry retinol as a non-covalently bound ligand in vivo6,7. It has a lower molecular weight and a different fluorescence excitation spectrum6 than that reported for the intensively studied retinol binding protein of serum8, demonstrating that we are dealing with a different protein. We have purified this cellular binding protein 1,000-fold from rat testes6 and 600-fold from rabbit lung7 and report here its specificity in binding various compounds exhibiting vitamin A activity in vivo.

Calcium and phosphorus in diet therapy of uremia

Significant advances have been made in understanding the pathogenesis of renal osteodystrophy. Several factors play a role in the development of uremic bone disease, such as secondary hyperparathyroidism, PTH resistance at the level of bone, abnormalities in vitamin D metabolism, and factors contributing to extraosseous calcification. Through a better understanding of the pathogenetic mechanisms, a more rational approach to the dietary management of patients with chronic renal failure has been possible. In view of present knowledge, the following preventive and therapeutic interventions can be recommended: provision of liberal calcium intake in the diet, appropriate restriction of phosphorus intake, provision of potent vitamin D analogs, and close attention to the calcium-phosphorus product. With careful attention to these recommendations, the bone lesions of secondary hyperparathyroidism and osteomalacia may be minimized and even prevented.