Inhibition of vitamin D metabolism by ethane-1-hydroxyl-1, 1-diphosphonate

The administration of disodium-ethane-1-hydroxy-1,1-diphosphonate (20 mg/kg body weight subcutaneously) to chicks given adequate amounts of vitamin D₃ causes a hypercalcemia, inhibits bone mineralization, and inhibits intestinal calcium transport. The administration of 1,25-dihydroxyvitamin D₃, a metabolically active form of vitamin D₃, restores intestinal calcium absorption to normal but does not restore bone mineralization in disodium-ethane-1-hydroxy-1,1-diphosphonate-treated chicks. In rachitic chicks, the disodium-ethane-1-hydroxy-1,1-diphosphonate treatment does not further reduce the low intestinal calcium transport values while it nevertheless further reduces bone ash levels and increases serum calcium concentration.These observations prompted a more detailed study of the relationship between disodium-ethane-1-hydroxy-1,1-diphosphonate treatment and vitamin D metabolism. A study of the hydroxylation of 25-hydroxyvitamin D₃ in an in vitro system employing kidney mitochondria from chicks receiving disodium-ethane-1-hydroxy-1,1-diphosphonate treatment demonstrates a marked decrease in 1,25-dihydroxyvitamin D₃ production and a marked increase in the 24,25-dihydroxyvitamin D₃ production. In addition, the in vivo metabolism of 25-hydroxy-[26,27-³H]vitamin D₃ in disodium-ethane-1-hydroxy-1,1-diphosphonate treated chicks supports the in vitro observations. In rachitic chicks the disodium-ethane-1-hydroxy-1,1-diphosphonate treatment markedly reduces the 25-hydroxyvitamin D₃-1-hydroxylase activity of kidney, but does not increase the 25-hydroxyvitamin D₃-24-hydroxylase.These results provide strong evidence that large doses of disodium-ethane-1-hydroxy-1,1-diphosphonate produce a marked effect on calcium metabolism via alterations in the metabolism of vitamin D as well as the expected direct effect on the bone.     

Pharmacological induction of ferritin prevents osteoblastic transformation of smooth muscle cells

Vascular calcification is a frequent complication of atherosclerosis, diabetes and chronic kidney disease. In the latter group of patients, calcification is commonly seen in tunica media where smooth muscle cells (SMC) undergo osteoblastic transformation. Risk factors such as elevated phosphorus levels and vitamin D₃ analogues have been identified. In the light of earlier observations by our group and others, we sought to inhibit SMC calcification via induction of ferritin. Human aortic SMC were cultured using β‐glycerophosphate with activated vitamin D₃, or inorganic phosphate with calcium, and induction of alkaline phosphatase (ALP) and osteocalcin as well as accumulation of calcium were used to monitor osteoblastic transformation. In addition, to examine the role of vitamin D₃ analogues, plasma samples from patients on haemodialysis who had received calcitriol or paricalcitol were tested for their tendency to induce calcification of SMC. Addition of exogenous ferritin mitigates the transformation of SMC into osteoblast‐like cells. Importantly, pharmacological induction of heavy chain ferritin by 3H‐1,2‐Dithiole‐3‐thione was able to inhibit the SMC transition into osteoblast‐like cells and calcification of extracellular matrix. Plasma samples collected from patients after the administration of activated vitamin D₃ caused significantly increased ALP activity in SMC compared to the samples drawn prior to activated vitamin D₃ and here, again induction of ferritin diminished the osteoblastic transformation. Our data suggests that pharmacological induction of ferritin prevents osteoblastic transformation of SMC. Hence, utilization of such agents that will cause enhanced ferritin synthesis may have important clinical applications in prevention of vascular calcification.     

The isolation and identification of vitamin D2 and vitamin D3 from Medicago sativa (alfalfa plant)

Vitamin D₂ and vitamin D₃ were isolated from Medicago sativa (alfalfa) grown under field and laboratory conditions and then irradiated with ultraviolet light. The vitamins were identified by ultraviolet absorption, mass spectroscopy, and comparison with synthetic standards on several chromatographic systems. Sun-cured, field-grown alfalfa contained vitamin D₂ at a concentration of 48 ng/g (1920 IU/kg) and vitamin D₃ at 0.63 ng/g (25 IU/kg). Laboratory-grown alfalfa, artificially irradiated, contained vitamin D₂ at a concentration of 80 ng/g and vitamin D₃ at 1.0 ng/g. Therefore, the presence of vitamin D₂, as well as vitamin D₃, has unequivocally been demonstrated in alfalfa plant tissue.     

Metabolism of 25-hydroxyvitamin D3 in kidney homogenates of chicks supplemented with vitamin D3.

Kidney homogenates from chicks fed a vitamin D-deficient diet for 10 days and supplemented with 6.5 nmol of vitamin D₃ 48 hr prior to sacrifice metabolized $\text{in}\text{vitro}\text{[}{}^3\text{H}\text{]}$-25-hydroxyvitamin D₃ (25-OH-D₃) to 24,25-dihydroxyvitamin D₃ [24,25-(OH)₂-D₃] and 3 other metabolites (peaks A, C and E). When the homogenates were incubated with purified [³H]-24,25-(OH)₂-D₃, 3 similar metabolites (peaks A′, C′ and E′) were produced. On high pressure liquid chromatography, peaks A, C and E migrated to exactly the same respective positions as peaks A′, C′ and E′. Kidney homogenates from D-deficient chicks failed to produce these metabolites from [³H]-25-OH-D₃ or [³H]-24,25-(OH)₂-D₃. These results strongly suggest that the new metabolites reported here are synthesized via 24,25-(OH)₂-D₃ in the kidney of chicks supplemented with vitamin D₃.     

Quantifying the vitamin D3 synthesizing potential of UVB lamps at specific distances over time

The purpose of this study was to quantify the ultraviolet B (UVB) output and in vitro previtamin D₃ synthesis over time from various artificial light sources. Three incandescent lamps, T‐Rex Active UVHeat 160 watt spot, T‐Rex Active UVHeat 160 watt flood, and ZooMed PowerSun 160 watt flood, and two 1.2 m fluorescent lamps, Sylvania Blacklight 350 BL and ZooMed Reptisun 5.0, were studied. Total UVB irradiance and concentration of previtamin D synthesized using an in vitro ampoule model were quantified initially and at monthly intervals for 1 year. Incandescent lamps were measured at distances of 0.9 and 1.5 m while fluorescent lamps were measured at distances of 30.5 and 45.7 cm at the lamp's center, using both the radiometer and ampoules. Fluorescent lamp irradiance was also measured at the lamp's ends. Data were analyzed as a repeated measures split‐plot in time using SAS with all mean differences determined using Least Squares Means. Incandescent lamp irradiance differences were seen at various distances. The UVHeat lamps had consistently higher previtamin D₃ production and irradiance readings compared with the PowerSun lamp. Reptisun 5.0 was consistently higher in UVB irradiance over Sylvania BL 350 at both 30.5 and 45.7 cm. However, there were no differences when comparing conversion of 7‐dehydrocholesterol to previtamin D₃. Irradiance differences were detected between the centers and ends of the fluorescent lamps. Until UVB requirements for vitamin D₃ synthesis in animals are determined, it is impossible to state that one light is superior to another. Zoo Biol 29:741–752, 2010. © 2010 Wiley‐Liss, Inc.     

Intestinal Na/Ca exchanger protein and gene expression are regulated by 1,25(OH)2D3 in vitamin D-deficient chicks

The role of 1,25(OH)₂D₃ on the intestinal NCX activity was studied in vitamin D-deficient chicks (-D) as well as the hormone effect on NCX1 protein and gene expression and the potential molecular mechanisms underlying the responses. Normal, -D and -D chicks treated with cholecalciferol or 1,25(OH)₂D₃ were employed. In some experiments, -D chicks were injected with cycloheximide or with cycloheximide and 1,25(OH)₂D₃ simultaneously. NCX activity was decreased by -D diet, returning to normal values after 50 IU daily of cholecalciferol/10 days or a dose of 1 μg calcitriol/kg of b.w. for 15 h. Cycloheximide blocked NCX activity enhancement produced by 1,25(OH)₂D₃. NCX1 protein and gene expression were diminished by -D diet and enhanced by 1,25(OH)₂D₃. Vitamin D receptor expression was decreased by -D diet, effect that disappeared after 1,25(OH)₂D₃ treatment. Rapid effects of 1,25(OH)₂D₃ on intestinal NCX activity were also demonstrated. The abolition of the rapid effects through addition of Rp-cAMPS and staurosporine suggests that non genomic effects of 1,25(OH)₂D₃ on NCX activity are mediated by activation of PKA and PKC pathways. In conclusion, 1,25(OH)₂D₃ enhances the intestinal NCX activity in -D chicks through genomic and non genomic mechanisms.     

UVB Exposure of Farm Animals: Study on a Food-Based Strategy to Bridge the Gap between Current Vitamin D Intakes and Dietary Targets

Vitamin D deficiency is a global health problem. This study aimed to investigate the efficacy of ultraviolet (UV) B radiation for improving vitamin D₃ content of eggs and meat. In a two-factorial design hens that received diets with 0 (-D₃) or 3,000 IU (+D₃) vitamin D₃/kg were non-exposed (-UVB) or exposed to UVB radiation (+UVB) for 3 h daily over 4 weeks. Data show that UVB radiation was very effective in raising the vitamin D₃ content of egg yolk and meat. Egg yolk from +UVB/−D₃ hens had a higher vitamin D₃ content (17.5±7.2 µg/100 g dry matter (DM)) than those from the –UVB/+D₃ group (5.2±2.4 µg/100 g DM, p<0.01). Vitamin D₃ content in egg yolk of vitamin D₃-supplemented hens could be further increased by UVB radiation (32.4±10.9 µg/100 g DM). The content of 25-hydroxyvitamin D₃ (25(OH)D₃) in the egg yolk also increased in response to UVB, although less pronounced than vitamin D₃. Meat revealed about 4-fold higher vitamin D₃ contents in response to UVB than to dietary vitamin D₃ (p<0.001). In conclusion, exposure of hens to UVB is an efficient approach to provide consumers with vitamin D₃-enriched foods from animal sources.     

Evidence for aqueous soluble vitamin D-like substances in the calcinogenic plant, Tristetum flavescens

A crude aqueous extract of the leaves of $\text{T. flavescens}$ when administered orally to vitamin D-deficient chicks produced significant increases in plasma phosphate but had little effect on plasma calcium. When chicks, fed a high strontium diet to inhibit endogenous 1,25(OH)₂ vitamin D₃ production and intestinal calcium transport, were dosed with the extract or synthetic 1,25(OH)₂D₃⁴⁵Ca absorption from the duodenum $\text{in vivo}$ was stimulated, whereas vitamin D₃ was ineffective. Partial purification of the crude extract on a Sephadex GH25 column yielded two factors, one of which mimicked 1,25 (OH)₂D₃ activity in chicks fed the high strontium diet while the other produced a significant increase in plasma phosphate. The presence of these substances, together with previously demonstrated organic solvent soluble vitamin D-type activity, may account for the calcinogenic nature of the plant.     

Effect of alcohol on renal vitamin D metabolism in chickens.

Intraperitoneal administration of ethanol to young chickens (both vitamin D-replete and vitamin D-deficient) produced a significant impairment of renal 25 hydroxyvitamin D₃ 1α-hydroxylase (EC 1.14.13.13) activity with no significant change in serum calcium or phosphorus. In ethanol treated D-replete chicks the renal 25 hydroxyvitamin D₃ 24-hydroxylase activity was enhanced, and serum 25 hydroxyvitamin D₃ was significantly increased. The alkaline phosphatase levels in the D-deficient ethanol treated chicks were significantly less than the controls. Our data suggest that the impairment of the metabolic effects of vitamin D due to ethanol occurs chiefly via a renal, rather than a hepatic mechanism. Furthermore, 1α -hydroxylated metabolites of vitamin D would appear to be the logical treatment of choice for the bone disease of alcoholism.     

Biotransformations of 25-hydroxyvitamin D3 by kidney microsomes.

Vitamin D₃-deficient chick kidney microsomes $\text{in}\text{vitro}$ metabolize 25-hydroxy-[26(27)-methyl-³H]-vitamin D₃ to yet structurally unidentified polar metabolites previously designated MIC-I and MIC-II. Kidney microsomes of vitamin D₃-repleted chicks could not be demonstrated to produce these metabolites when ³H was the radioactive isotope in positions C-26 and C-27 of the substrate. However, when 25-hydroxy-[26,27-¹⁴C]-vitamin D₃ was the radioactive substrate, MIC-I and MIC-II production was independent of the vitamin D₃ status of the chicks. These results suggest that under conditions of vitamin D₃-sufficiency, there is augmented sequential kidney metabolism of 25-hydroxyvitamin D₃ to products with modified side-chains involving C-26 and/or C-27. It is possible that this metabolism is responsible for the regulation of kidney cellular concentrations of 25-hydroxyvitamin D₃.     

Responses of Corpuscles of Stannius to intra-peritoneal vitamin-D3 administration in teleost Labeo rohita (Hamilton, 1822) reared in water with two different levels of calcium concentration

This study assessed the responses of vitamin-D₃ intraperitoneally injected to Rohu, Labeo rohita @ of 0 IU/kg bw (only solvent), 100 IU/kg bw and 500 IU/kg bw reared in 20 and 40 ppm of calcium (Ca) enriched water. The cellular changes in Corpuscles of Stannius (CS) gland, serum Ca, and inorganic phosphate (P_i) level were analysed up to the 60th day. Rohu administered with 100 IU/kg bw D₃ and exposed to 40 ppm Ca-rich water exhibited notable hyperplasia of CS compared with their control groups. Notable changes with high serum Ca level (13.87 ± 0.3 mg/dl) was detected on the 5th day in fish exposed to 40 ppm Ca-rich water, while related values attained (13.74 ± 0.1 mg/dl) only after 7 days in 20 ppm Ca-rich water of 500 IU/kg bw vitamin D₃ injection. Similarly, high serum P_i level (7.66 ± 0.2 mg/dl) in 40 ppm Ca injected with D₃ at 500 IU/kg bw. The results demonstrated that the Ca homeostasis of Labeo rohita is influenced by intra-peritoneal vitamin D₃. Progressive studies should be conducted by increasing the dose of vitamin D₃ to investigate optimum dose/supplement in feed for commercially important aquaculture teleost Labeo rohita for maximum and sustainable absorption of Ca from the variable water Calcium levels to maintain Ca²⁺ homeostasis.     

Synthesis of vitamin D5: its biological activity relative to vitamins D3 and D2.

The chemical synthesis, spectral characterization, and biological activity of vitamin D₅ in vitamin D-deficient rats is reported. Vitamin D₅ is about 180-fold less active than vitamin D₃ in calcification of rachitic cartilage and about 100- to 200-fold less active in induction of bone-calcium mobilization. In stimulation of intestinal-calcium transport, vitamin D₅ is about 80-fold less active than vitamin D₃. Vitamins D₂ and D₃ appear to be equiactive in all three responses when low doses are administered.     

Kidney microsomal metabolism of 25-hydroxyvitamin D3+.

Vitamin D₃-deficient chick kidney microsomes $\text{in vitro}$ metabolize 25-hydroxyvitamin D₃ to two polar metabolites by a pathway which may involve side-chain modification. Molecular oxygen and a source of reduced nicotinamide adenine dinucleotide phosphate are required for this metabolism. Kidney cytosol obtained from deficient chicks or kidney microsomes of vitamin D₃-repleted chicks do not metabolize 25-hydroxyvitamin D₃. The two products are tentatively designated MIC-I and MIC-II.     

Evaluation of vitamin D3 metabolites in Callithrix jacchus (common marmoset)

Vitamin D₃ (cholecalciferol) is endogenously produced in the skin of primates when exposed to the appropriate wavelengths of ultraviolet light (UV-B). Common marmosets (Callithrix jacchus) maintained indoors require dietary provision of vitamin D₃ due to lack of sunlight exposure. The minimum dietary vitamin D₃ requirement and the maximum amount of vitamin D₃ that can be metabolized by marmosets is unknown. Observations of metabolic bone disease and gastrointestinal malabsorption have led to wide variation in dietary vitamin D₃ provision amongst research institutions, with resulting variation in circulating 25-hydroxyvitamin D₃ (25(OH)D₃), the accepted marker for vitamin D sufficiency/deficiency. Multiple studies have reported serum 25(OH)D₃ in captive marmosets, but 25(OH)D₃ is not the final product of vitamin D₃ metabolism. In addition to serum 25(OH)D_3, we measured the most physiologically active metabolite, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), and the less well understood metabolite, 24,25-dihydroxyvitamin D₃ (24,25(OH)₂D₃) to characterize the marmoset's ability to metabolize dietary vitamin D₃. We present vitamin D₃ metabolite and related serum chemistry value colony reference ranges in marmosets provided diets with 26,367 (Colony A, N = 113) or 8,888 (Colony B, N = 52) international units (IU) of dietary vitamin D₃ per kilogram of dry matter. Colony A marmosets had higher serum 25(OH)D₃ (426 ng/ml [SD 200] vs. 215 ng/ml [SD 113]) and 24,25(OH)₂D₃ (53 ng/ml [SD 35] vs. 7 ng/ml [SD 5]). There was no difference in serum 1,25(OH)₂D₃ between the colonies. Serum 1,25(OH)₂D₃ increased and 25(OH)D₃ decreased with age, but the effect was weak. Marmosets tightly regulate metabolism of dietary vitamin D₃ into the active metabolite 1,25(OH)₂D₃; excess 25(OH)D₃ is metabolized into 24,25(OH)₂D₃. This ability explains the tolerance of high levels of dietary vitamin D₃ by marmosets, however, our data suggest that these high dietary levels are not required.     

Calcium lysosomes in rachitic and vitamin D3 replete chick duodenal absorptive cells

Calcium-containing lysosomes were described in a previous communication in this series (Davis et al., 1979). Their potential role in intestinal calcium uptake and transcellular transport was hypothesized. To further this notion, the effects of a rachitogenic diet and vitamin D₃ repletion were investigated. Intestinal absorptive cells from chicks maintained on a vitamin D deficient diet were characterized by decreased numbers of supranuclear calcium lysosomes. In contrast, intestinal cells from chicks given vitamin D₃ (cholecalciferol) subsequent to the rachitogenic diet showed numerous large compound supranuclear calcium lysosomes. Since other steroid hormones are known to effect lysosomes, it is tempting to speculate that vitamin D, itself a steroid hormone, may activate lysosomes which themselves might be involved in calcium homeostatic mechanisms.     

Studies on the mode of action of calciferol. X. 24-Nor-25-hydroxyvitamin D3, an analog of 25-hydroxyvitamin D3 having "anti-vitamin" activity.

24-Nor-25-hydroxyvitamin D₃, an analog of 25-hydroxyvitamin D₃, has been chemically synthesized in six steps. This steroid was tested in chicks, $\text{in vivo}$, for its ability to generate the classic vitamin D mediated responses of stimulation of intestinal calcium transport and bone calcium mobilization. Although the 24-nor-25-OH-vitamin D₃ itself exhibited no biological activity in these assays, the analog was found to inhibit the normal responses produced by a physiological dose of vitamin D₃. These results suggest that 24-nor-25-OH-vitamin D₃ may satisfy certain requirements expected of a calciferol “anti-vitamin.”     

Vitamin D-dependent calcium-binding protein synthesis by chick kidney and duodenal polysomes

The hormonally active form of vitamin D, 1,25-dihydroxy vitamin D₃, is known to induce in the intestine and kidney of chicks the synthesis of a calcium-binding protein (CaBP). Here we report a correlation between the tissue levels of CaBP and the levels of apparent messenger RNA in total polysomes as determined by the vitamin D and dietary calcium status. Polysomes from pooled duodenal mucosa and kidney were prepared by the Mg²⁺ precipitation method. After translation in a heterologous, rabbit nuclease-treated reticulocyte system, the immunoprecipitated pellet of CaBP was dissolved and the proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gels. When 13 nmol of D₃ was given to 4-week-old rachitic chicks which were sacrificed 48 h later, it was found that the duodenum had eightfold more apparent mRNA for CaBP in the polysomes than the kidney. This was also reflected in the values of CaBP/mg protein in these tissues (duodenum, 7 μg/mg vs kidney, 0.9 μ/mg). Also, after giving D₃, there was a twofold increase in both apparent mRNA levels in the polysomes and in CaBP levels in the duodena of chicks which were raised on low-calcium diets versus chicks raised on high-calcium diets. While apparent mRNA for CaBP was present in polysomes from rachitic chick kidney, it was not detectable in the duodenum. From these studies it appears that the induction of CaBP by 1,25(OH)₂D₃ in both the intestine and kidney is determined by similar control mechanisms.     

Isolation,identification, and biological activity of 23,25,26-trihydroxyvitamin D3, an in vitro and in vivo metabolite of vitamin D3

Kidney homogenates from vitamin D₃-supplemented chicks incubated with 25-hydroxyvitamin D₃ [25(OH)D₃] produce significant quantities of a new, unknown vitamin D metabolite. This metabolite was isolated in pure form from such incubation mixtures by using Sephadex LH-20 column chromatography followed by high-pressure liquid chromatography. This metabolite has been identified as 23,25,26-trihydroxyvitamin D₃ [23,25,26(OH)₃D₃] by loss of radioactivity from 25-hydroxy[23,24-³H]vitamin D₃ and 25-hydroxy-[26,27-methyl-³H]vitamin D₃, ultraviolet absorption spectrophotometry, mass spectrometry, and periodate cleavage oxidation followed by mass spectrometry. This same metabolite was also isolated from the serum of rats given large doses of vitamin D₃, and structurally characterized as 23,25,26-trihydroxyvitamin D₃. As yet, the stereochemistry at the C-23 and C-25 positions of the natural product remains unknown. A comparison of responses to a single dose level (500 ng) of 23,25,26(OH)₃D₃ or 25(OH)D₃ over 96 h in vitamin D-deficient rats indicated that the new metabolite had no capability to mediate bone calcium mobilization and that it was only weakly active in stimulating intestinal calcium transport.     

Differencesin-Vitamin-D3-Dosing-Regimens in a Geriatric Community-Dwelling Population

ObjectiveThe adequate dose of vitamin D supple mentation for community-dwelling elderly people has not been thoroughly investigated. This study aims to determine the efficacy of a low-dose and a higher dose of vitamin D₃ in maintaining 25-hydroxyvitamin D [25(OH)D] levels at or above 30 ng/mL.MethodsThis was a single site, double-blind, ran domized exploratory clinical trial that enrolled adults 65 years of age and older. Within strata of baseline 25(OH) D levels (< 30 versus ≥ 30 ng/mL) subjects were random ized in a 1:2 ratio to receive either 400 or 2,000 IU vitamin D₃ daily for 6 months. The main outcome measures were changes in serum 25(OH)D levels according to baseline 25(OH)D levels and dose of vitamin D3.ResultsAt baseline, 41 of 105 participants (39%) had low 25(OH)D levels (< 30 ng/mL). After 6 months of vitamin D₃ supplementation, 21 of 32 participants (66%) receiving 400 IU and 14 of 59 participants (24%) receiving 2,000 IU of vitamin D₃ still had low 25(OH)D levels. Thelargest increases in serum 25(OH)D levels were observed in subjects with baseline levels < 30 ng/mL who received 2,000 IU of vitamin D daily.ConclusionRegardless of baseline 25(OH)D level, in persons 65 years of age and older, 6-month vitamin D₃ supplementation with 400 IU daily resulted in low 25(OH) D in most individuals, while 2,000 IU daily maintained 25(OH)D levels within an acceptable range in most people on this regimen. (Endocr Pract. 2012;18:847-854)     

Occurrence of cholecalciferol in the calcinogenic plant Trisetum flavescens.

The grass Trisetum flavescens causes severe calcification of soft tissues upon ingestion by various species, which has been ascribed by others to a 1,25(OH)₂ vitamin D₃-like activity.By a special purification procedure involving high pressure liquid chromatography and continuous biological testing the active principle was purified. By means of $\text{GC}\text{MS}$ it was identified as cholecalciferol, being present in a concentration of about 0.1 ppm in the lyophylized plant dry matter. 1,25(OH)₂ vitamin D₃ or other metabolites of vitamin D₃ were not present. Since such low concentrations could hardly explain the calcinosis observed, a more active “bound form” of vitamin D₃ may be present in Trisetum flavescens.