A comparison of UVb compact lamps in enabling cutaneous vitamin D synthesis in growing bearded dragons.

The effect of exposure to different UVb compact lamps on the vitamin D status of growing bearded dragons (Pogona vitticeps) was studied. Forty-two newly hatched bearded dragons (<24 h old) were allocated to six treatment groups (n = 7 per group). Five groups were exposed to different UVb compact lamps for two hours per day, with a control group not exposed to UVb radiation. At 120 days of age, blood samples were obtained and concentrations of 25(OH)D3 , Ca, P and uric acid were determined. In addition, plasma 25(OH)D3 concentration was determined in free-living adult bearded dragons to provide a reference level. Only one treatment resulted in elevated levels of 25(OH)D3 compared to the control group (41.0 ± 12.85 vs. 2.0 ± 0.0 nmol/L). All UVb-exposed groups had low 25(OH)D3 plasma levels compared to earlier studies on captive bearded dragons as well as in comparison with the free-living adult bearded dragons (409 ± 56 nmol/L). Spectral analysis indicated that all treatment lamps emitted UVb wavelengths effective for some cutaneous vitamin D synthesis. None of these lamps, under this regime, appeared to have provided a sufficient UVb dose to enable synthesis of plasma 25(OH)D3 levels similar to those of free-living bearded dragons in their native habitat.

Bioconversion of vitamin D3 to calcifediol by using resting cells of Pseudonocardia sp.

OBJECTIVES: Resting cells of Pseudonocardia sp. KCTC 1029BP were used for the bioconversion of vitamin D3 to calcifediol which is widely used to treat osteomalacia and is industrially produced by chemical synthesis. RESULTS: To obtain the maximum bioconversion yield of calcifediol by the microbial conversion of vitamin D3, a two-step optimization process was used, including the Plackett-Burman and the central composite designs. Six variables, namely agitation speed, aeration rate, resting cell concentration, vitamin D3 concentration, temperature, and pH, were monitored. Of these, aeration rate, resting cell concentration, and temperature were selected as key variables for calcifediol production and were optimized using the central composite design. Optimal bioconversion conditions obtained were as follows: aeration rate of 0.2 vvm, resting cell concentration of 4.7% w/v, and temperature of 33 °C. CONCLUSION: Using the optimal conditions, 356 mg calcifediol l(-1) was obtained with a bioconversion yield of 59.4% in a 75 l fermentor. These are the highest values reported to date.

[Biosynthesis of calcifediol and calcitriol: a review].

VD3 is a crucial vitamin for human health, as it enhances calcium absorption in the intestines and prevent rickets. Calcifediol (25(OH)VD3) and calcitriol (1α,25(OH)2VD3) are two derivatives of vitamin D3 that play an important role in preventing and treating osteoporosis, as well as regulating human physiological functions. Currently, the production of calcifediol, and calcitriol primarily relies on chemical synthesis, which has disadvantages such as low product yield, numerous by-products, and environmental unfriendliness. Therefore, developing a green, safe, and environmentally friendly biocatalytic synthesis pathway is of utmost importance. This article mainly reviews the biocatalytic synthesis pathways of calcifediol, and calcitriol. The P450 enzymes, including P450 monooxygenases (cytochrome P450 monooxygenases, CYPs) and P450 peroxygenases (unspecific peroxygenases, UPOs), are crucial for the production of calcifediol and calcitriol. The catalytic mechanism of the extensively studied P450 monooxygenases, the selection of suitable redox partners, and the key residues involved in the enzyme's catalytic activity are analyzed. In addition, the review explores H2O2-driven UPOs, including their catalytic mechanism, strategies for high heterologous expression, and in situ regeneration of H2O2. UPOs are regarded as highly promising biocatalysts because they can facilitate reactions without the need for expensive cofactors and redox partners. This review offers insights into the engineering of P450 for the efficient production of vitamin D3 derivatives.

A single mutation at the ferredoxin binding site of P450 Vdh enables efficient biocatalytic production of 25-hydroxyvitamin D(3).

Vitamin D3 hydroxylase (Vdh) from Pseudonocardia autotrophica is a cytochrome P450 monooxygenase that catalyzes the two-step hydroxylation of vitamin D3 (VD3 ) to produce 25-hydroxyvitamin D3 (25(OH)VD3 ) and 1α,25-dihydroxyvitamin D3 (1α,25(OH)2 VD3 ). These hydroxylated forms of VD3 are useful as pharmaceuticals for the treatment of conditions associated with VD3 deficiency and VD3 metabolic disorder. Herein, we describe the creation of a highly active T107A mutant of Vdh by engineering the putative ferredoxin-binding site. Crystallographic and kinetic analyses indicate that the T107A mutation results in conformational change from an open to a closed state, thereby increasing the binding affinity with ferredoxin. We also report the efficient biocatalytic synthesis of 25(OH)VD3 , a promising intermediate for the synthesis of various hydroxylated VD3 derivatives, by using nisin-treated Rhodococcus erythropolis cells containing VdhT107A . The gene-expression cassette encoding Bacillus megaterium glucose dehydrogenase-IV was inserted into the R. erythropolis chromosome and expressed to avoid exhaustion of NADH in a cytoplasm during bioconversion. As a result, approximately 573 µg mL(-1) 25(OH)VD3 was successfully produced by a 2 h bioconversion.

Permeabilization induced by lipid II-targeting lantibiotic nisin and its effect on the bioconversion of vitamin D3 to 25-hydroxyvitamin D3 by Rhodococcus erythropolis.

Vitamin D3 (VD3) is a fat-soluble prohormone in mammals. VD3 is inert and must be activated by hydroxylation at the C-25 and C-1α positions to exert its biological activity. We recently accomplished the bioconversion of VD3 to 25(OH)VD3 with a recombinant strain of Rhodococcus erythropolis and found that the permeability of VD3 into the cytoplasm may be the rate-limiting step of 25(OH)VD3 production (Sallam et al., 2010). When the cells were treated with the lipid II-targeting lantibiotic nisin, the permeability of green chemiluminescent cyclodextrin (GCCD), which is used as a model substrate instead of VD3-partially methylated-ß-cyclodextrin (PMCD) complex, was drastically induced. Nisin also induced VD3 hydroxylation, and the rate was correlated with the expression levels of Vdh and its redox partner proteins. In the bioconversion reaction, the stability of the redox partner proteins and the additional NADH-regenerating system are crucial for VD3 hydroxylation. The degradation rate of the [2Fe-2S] cluster of ferredoxin ThcC from R. erythropolis NI86/21 is faster than that of AciB from Acinetobacter sp. OC4. Therefore, the nisin-treated R. erythropolis cells coexpressing Vdh and AciBC (1176.5 µg) exhibited much greater 25(OH)VD3 production than the cells coexpressing Vdh and ThcCD (431.7 µg) after four consecutive 16 h reactions. These results suggest that nisin forms nisin-lipid II pore complexes in the Rhodococcus membrane that increase the accessibility of VD3-PMCD complexes to the inside of the cells. Furthermore, nisin-treated Rhodococcus cells can be utilized for the bioconversion of other fat-soluble chemicals.

Interdependence between body surface area and ultraviolet B dose in vitamin D production: a randomized controlled trial.

BACKGROUND: Ultraviolet (UV) B radiation increases serum vitamin D level expressed as 25-hydroxyvitamin-D(3) [25(OH)D], but the relationship to body surface area and UVB dose needs investigation. OBJECTIVE: To investigate the importance of body surface area and UVB dose on vitamin D production after UVB exposure. METHODS: We randomized 92 participants to have 6%, 12% or 24% of their skin exposed to 0·75 (7·5 mJ cm(-2) at 298 nm using the CIE erythema action spectrum), 1·5 (15 mJ cm(-2) ) or 3·0 (30 mJ cm(-2) ) standard erythema doses (SED) of UVB. Each participant underwent four UVB exposures at intervals of 2-3 days. Skin pigmentation and 25(OH)D levels were measured before and 48 h after the final exposure. RESULTS: The increase in 25(OH)D after irradiation [Δ25(OH)D] was positively correlated with body surface area (P = 0·006; R(2) = 0·08) and UVB dose (P < 0·0001; R(2) = 0·28), and negatively correlated with baseline 25(OH)D (P < 0·0001; R(2) = 0·18), for the entire data sample. However, when analysing each body surface area separately, we found a significant UVB response correlation for 6% (P < 0·0001; R(2) = 0·48) and 12% (P = 0·0004; R(2) = 0·35), but not for 24%. We also found a significant skin area response correlation for 0·75 SED (P < 0·0001; R(2) = 0·56), but not for 1·5 and 3·0 SED when analysing each UVB dose separately. The relationships did not change significantly after adjustment of Δ25(OH)D for baseline 25(OH)D. CONCLUSION: The increase in 25(OH)D depends mainly on the UVB dose; however, for small UVB doses the area of irradiated body surface is important.

Reduced hepatic synthesis of calcidiol in uremia.

Calcidiol insufficiency is highly prevalent in chronic kidney disease (CKD), but the reasons for this are incompletely understood. CKD associates with a decrease in liver cytochrome P450 (CYP450) enzymes, and specific CYP450 isoforms mediate vitamin D(3) C-25-hydroxylation, which forms calcidiol. Abnormal levels of parathyroid hormone (PTH), which also modulates liver CYP450, could also contribute to the decrease in liver CYP450 associated with CKD. Here, we evaluated the effects of PTH and uremia on liver CYP450 isoforms involved in calcidiol synthesis in rats. Uremic rats had 52% lower concentrations of serum calcidiol than control rats (P < 0.002). Compared with controls, uremic rats produced 71% less calcidiol and 48% less calcitriol after the administration of vitamin D(3) or 1alpha-hydroxyvitamin D(3), respectively, suggesting impaired C-25-hydroxylation of vitamin D(3). Furthermore, uremia associated with a reduction of liver CYP2C11, 2J3, 3A2, and 27A1. Parathyroidectomy prevented the uremia-associated decreases in calcidiol and liver CYP450 isoforms. In conclusion, these data suggest that uremia decreases calcidiol synthesis secondary to a PTH-mediated reduction in liver CYP450 isoforms.

Melanin has a Small Inhibitory Effect on Cutaneous Vitamin D Synthesis: A Comparison of Extreme Phenotypes.

Epidemiology suggests that melanin inhibits cutaneous vitamin D3 synthesis by UVR. Laboratory investigations assessing the impact of melanin on vitamin D production have produced contradictory results. We determined the effect of melanin on vitamin D3 photosynthesis in healthy young volunteers (n = 102) of Fitzpatrick skin types II-VI (white to black). Participants, irrespective of skin type, were exposed to the same suberythemal UVR dose, to 85% body surface area, using solar simulated UVR or narrowband UVB (311 nm). This was repeated five times with intervals of 3-4 days between UVR exposures. Blood was taken before, during, and after the irradiation and assessed for serum 25-hydroxyvitamin D3 (25[OH]D3) as a marker of vitamin D3 status. Linear UVR dose-dependent increases in 25(OH)D3 were highly significant (P ≤ 7.7 x 10-11). The ratios of regression slopes of the different skin type groups were compared, and only skin type II was significantly steeper than the other groups. Comparisons between extreme skin types II and VI showed melanin inhibition factors of approximately 1.3-1.4, depending on the UVR source. We conclude that the inhibitory effect of melanin on vitamin D3 synthesis is small, compared with erythema, but that this difference may be sufficient to explain the epidemiological data.

Glutathione deficiency induces epigenetic alterations of vitamin D metabolism genes in the livers of high-fat diet-fed obese mice.

Obesity has been correlating with low levels of glutathione (GSH) and 25-hydroxyvitamin D3 (25(OH)VD3). The liver is the principal site for the 25(OH)VD3 biosynthesis. This study investigated whether GSH deficiency induces epigenetic alterations that impair Vitamin D (VD) metabolism genes in the livers of HFD-fed mice. The expression of the VD metabolism genes CYP2R1 and CYP27A1 (25-hydroxylase), CYP27B1 (1-α-hydroxylase), and vitamin D receptor (VDR) were downregulated in the livers of mice fed an HFD (GSH- deficient) compared with control diet-fed group. The expression of CYP24A1 (24-hydroxylase) was significantly increased, which catabolizes both 25(OH)VD3 and 1α,25-hydroxyvitaminD3. Gene-specific hypermethylation of 25-hydroxylase, 1-α-hydroxylase, and VDR, and hypomethylation of CYP24A1 was observed in HFD-fed mice. GSH deficiency induced in cultured hepatocytes caused an increase in oxidative stress and alterations in VD regulatory genes. Similarly, elevated global DNA methylation, Dnmt activity, and 5-methylcytosine but decreased Tet activity and 5-hydroxymethylcytosine were observed in the GSH-deficient hepatocytes and the liver of HFD-fed mice. Replenishment of GSH by its prodrugs treatment beneficially altered epigenetic enzymes, and VD-metabolism genes in hepatocytes. HFD-induces GSH deficiency and epigenetically alters VD-biosynthesis pathway genes. This provides a biochemical mechanism for the VD-deficiency and potential benefits of GSH treatment in reducing 25(OH)VD3-deficiency.

1,25 Dihydroxyvitamin D increases hepatocyte cytosolic calcium levels. A potential regulator of vitamin D-25-hydroxylase.

1,25 dihydroxyvitamin D (1,25(OH)2D) has been demonstrated to inhibit hepatic 25 hydroxyvitamin D (25 OHD) production. Changes in cytosolic calcium have been shown to regulate cellular processes. Using the fluorescent dye Quin 2, we have investigated the effects of 1,25(OH)2D and 24,25(OH)2D on cytosolic calcium levels in hepatocytes. 1,25(OH)2D exposure for 5 min increases cytosolic calcium levels by 24% at a concentration of 100 pg/ml, 39% at a concentration of 1 ng/ml, and 50% at a concentration of 2 ng/ml. The latter increment occurs in both the presence and absence of extracellular calcium, indicating that 1,25(OH)2D is mobilizing intracellular calcium pools. 24,25(OH)2D, 10 ng/ml, does not increase cytosolic calcium levels while the calcium ionophore A23187, 3 microM, increases levels by 52%. Calcium inhibits hepatic 25 OHD synthesis in liver homogenates in a dose-dependent fashion, which can be prevented by chelation of calcium with EGTA. 1,25(OH)2D and A23187 decrease hepatocyte 25 OHD synthesis. The inhibitory effect of A23187 can be prevented by chelation of extracellular calcium. The data demonstrate that 1,25(OH)2D increases hepatocyte cytosolic calcium, and that these increments in cytosolic calcium may regulate some of the hepatic actions of the vitamin D metabolite.

Identification and regulation of 1,25-dihydroxyvitamin D3 receptor activity and biosynthesis of 1,25-dihydroxyvitamin D3. Studies in cultured bovine aortic endothelial cells and human dermal capillaries.

Because 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has been shown to play roles in both proliferation and differentiation of novel target cells, the potential expression of 1,25(OH)2D3 receptor (VDR) activity was investigated in cultured bovine aortic endothelial cells (BAEC). Receptor binding assays performed on nuclear extracts of BAEC revealed a single class of specific, high-affinity VDR that displayed a 4.5-fold increase in maximal ligand binding (Nmax) in rapidly proliferating BAEC compared with confluent, density-arrested cells. When confluent BAEC were incubated with activators of protein kinase C (PKC), Nmax increased 2.5-fold within 6-24 h and this upregulation was prevented by sphingosine, an inhibitor of PKC, as well as by actinomycin D or cycloheximide. Immunohistochemical visualization using a specific MAb disclosed nuclear localized VDR in venular and capillary endothelial cells of human skin biopsies, documenting the expression of VDR, in vivo, and validating the BAEC model. Finally, additional experiments indicated that BAEC formed the 1,25(OH)2D3 hormonal metabolite from 25(OH)D3 substrate, in vitro, and growth curves of BAEC maintained in the presence of 10(-8) M 1,25(OH)2D3 showed a 36% decrease in saturation density. These data provide evidence for the presence of a vitamin D microendocrine system in endothelial cells, consisting of the VDR and a 1 alpha-hydroxylase enzyme capable of producing 1,25(OH)2D3. That both components of this system are coordinately regulated, and that BAEC respond to the 1,25(OH)2D3 hormone by modulating growth kinetics, suggests the existence of a vitamin D autocrine loop in endothelium that may play a role in the development and/or functions of this pathophysiologically significant cell population.

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

Although ectopic expression of 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-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 1alpha-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 1alpha-OHase activity remains that found in some patients with granulomatous diseases where locally synthesized 1alpha,25(OH)(2)D(3) has the potential to spill-over into the general circulation. However, immunohistochemistry and mRNA analyses suggest that 1alpha-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)(2)D(3) 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)(2)D(3) in these cells and the functional significance of autocrine responses to 1alpha-hydroxylase. Data suggest that local synthesis of 1,25(OH)(2)D(3) may be a preferred mode of response to antigenic challenge in many tissues.

Effect of vitamin D-containing plant extracts on osteoporotic bone.

Adequate supply of vitamin D(3) is not sufficient for the prevention of post-menopausal osteoporosis, because of a tightly regulated critical step in formation of the most active vitamin D metabolite 1,25-dihydroxyvitamin D(3). Direct application of 1,25(OH)(2)D(3), however, was effective in reducing fracture rate and increasing bone mineral density as has been shown in large clinical studies. Extracts from Solanum glaucophyllum and Trisetum flavescens plants containing 1,25(OH)(2)D(3)-glycosides were characterized by their vitamin D-activity in a quail eggshell bioassay and applied in an osteoporosis model in ovariectomized rats. An extract from the grass T. flavescens and a purified extract from S. glaucophyllum were characterized by the absence of alkaloids and the analytically determined content of 1,25(OH)(2)D(3). In the ovariectomized rat model after 6 months duration, the bone metabolism relevant markers serum calcium, 1,25(OH)(2)D(3), urinary crosslinks and calcium were measured. At termination tibial mineral content was determined and as imaging procedure micro-computerized tomography was applied. The bisphosphonate alendronate was used as a positive standard. While alendronate reduced bone resorption, as seen in a reduced urinary crosslink excretion, both vitamin D metabolite-containing extracts were able to improve bone mineral density by an enhanced calcium turnover.

Characterization of cytochrome P450 CYP109E1 from Bacillus megaterium as a novel vitamin D3 hydroxylase.

In this study the ability of CYP109E1 from Bacillus megaterium to metabolize vitamin D3 (VD3) was investigated. In an in vitro system using bovine adrenodoxin reductase (AdR) and adrenodoxin (Adx4-108), VD3 was converted by CYP109E1 into several products. Furthermore, a whole-cell system in B. megaterium MS941 was established. The new system showed a conversion of 95% after 24h. By NMR analysis it was found that CYP109E1 catalyzes hydroxylation of VD3 at carbons C-24 and C-25, resulting in the formation of 24(S)-hydroxyvitamin D3 (24S(OH)VD3), 25-hydroxyvitamin D3 (25(OH)VD3) and 24S,25-dihydroxyvitamin D3 (24S,25(OH)2VD3). Through time dependent whole-cell conversion of VD3, we identified that the formation of 24S,25(OH)2VD3 by CYP109E1 is derived from VD3 via the intermediate 24S(OH)VD3. Moreover, using docking analysis and site-directed mutagenesis, we identified important active site residues capable of determining substrate specificity and regio-selectivity. HPLC analysis of the whole-cell conversion with the I85A-mutant revealed an increased selectivity towards 25-hydroxylation of VD3 compared with the wild type activity, resulting in an approximately 2-fold increase of 25(OH)VD3 production (45mgl-1day-1) compared to wild type (24.5mgl-1day-1).

Hydroxylation of 20-hydroxyvitamin D3 by human CYP3A4.

20S-Hydroxyvitamin D3 [20(OH)D3] is the biologically active major product of the action of CYP11A1 on vitamin D3 and is present in human plasma. 20(OH)D3 displays similar therapeutic properties to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], but without causing hypercalcaemia and therefore has potential for development as a therapeutic drug. CYP24A1, the kidney mitochondrial P450 involved in inactivation of 1,25(OH)2D3, can hydroxylate 20(OH)D3 at C24 and C25, with the products displaying more potent inhibition of melanoma cell proliferation than 20(OH)D3. CYP3A4 is the major drug-metabolising P450 in liver endoplasmic reticulum and can metabolise other active forms of vitamin D, so we examined its ability to metabolise 20(OH)D3. We found that CYP3A4 metabolises 20(OH)D3 to three major products, 20,24R-dihydroxyvitamin D3 [20,24R(OH)2D3], 20,24S-dihydroxyvitamin D3 [20,24S(OH)2D3] and 20,25-dihydroxyvitamin D3 [20,25(OH)2D3]. 20,24R(OH)2D3 and 20,24S(OH)2D3, but not 20,25(OH)2D3, were further metabolised to trihydroxyvitamin D3 products by CYP3A4 but with low catalytic efficiency. The same three primary products, 20,24R(OH)2D3, 20,24S(OH)2D3 and 20,25(OH)2D3, were observed for the metabolism of 20(OH)D3 by human liver microsomes, in which CYP3A4 is a major CYP isoform present. Addition of CYP3A family-specific inhibitors, troleandomycin and azamulin, almost completely inhibited production of 20,24R(OH)2D3, 20,24S(OH)2D3 and 20,25(OH)2D3 by human liver microsomes, further supporting that CYP3A4 plays the major role in 20(OH)D3 metabolism by microsomes. Since both 20,24R(OH)2D3 and 20,25(OH)2D3 have previously been shown to display enhanced biological activity in inhibiting melanoma cell proliferation, our results show that CYP3A4 further activates, rather than inactivates, 20(OH)D3.

Tumoral Vitamin D Synthesis by CYP27B1 1-α-Hydroxylase Delays Mammary Tumor Progression in the PyMT-MMTV Mouse Model and Its Action Involves NF-κB Modulation.

Biologically active vitamin D (1,25-dihydroxycholecalciferol or 1,25(OH)2D) is synthetized from inactive prohormone 25-hydroxycholecalciferol (25(OH)D) by the enzyme CYP27B1 1-α-hydroxylase in kidney and several extrarenal tissues including breast. Although the development of breast cancer has been linked to inadequate vitamin D status, the importance of bioactive vitamin D production within tumors themselves is not fully understood. To investigate the role of tumoral vitamin D production in mammary epithelial cell progression to breast cancer, we conducted a Cre-loxP-mediated Cyp27b1 gene ablation in the mammary epithelium of the polyoma middle T antigen-mouse mammary tumor virus (PyMT-MMTV) mouse breast cancer model. Targeted ablation of Cyp27b1 was accompanied by significant acceleration in initiation of spontaneous mammary tumorigenesis. In vivo, cell proliferation, angiogenesis, cell cycle progression, and survival markers were up-regulated in tumors by Cyp27b1 ablation, and apoptosis was decreased. AK thymoma (AKT) phosphorylation and expression of several components of nuclear factor κB (NF-κB), integrin, and signal transducer and activator of transcription 3 (STAT3) signaling pathways were increased in Cyp27b1-ablated tumors compared with nonablated controls. In vitro, 1,25(OH)2D treatment induced a strong antiproliferative action on tumor cells from both ablated and nonablated mice, accompanied by rapid disappearance of NF-κB p65 from the nucleus and segregation in the cytoplasm. In contrast, treatment with the metabolic precursor 25(OH)D was only effective against cells from nonablated mice. 25(OH)D did not inhibit growth of Cyp27b1-ablated cells, and their nuclear NF-κB p65 remained abundant. Our findings demonstrate that in-tumor CYP27B1 1-α-hydroxylase activity plays a crucial role in controlling early oncogene-mediated mammary carcinogenesis events, at least in part by modulating tumoral cell NF-κB p65 nuclear translocation.

Cloning and nucleotide sequence of a bacterial cytochrome P-450VD25 gene encoding vitamin D-3 25-hydroxylase.

The gene encoding an enzyme that catalyzes the hydroxylation at position 25 of vitamin D-3 was cloned from an actinomycete strain, Amycolata autotrophica, by use of a host-vector system of Streptomyces lividans. The amino acid sequence deduced from the nucleotide sequence revealed that this enzyme, tentatively named P-450VD25, contains several regions of strong similarity with amino acid sequences of cytochromes P-450 from a variety of organisms, primarily in the regions of an oxygen-binding site and a heme ligand pocket. Especially, P-450VD25 shows end-to-end similarity in amino acid sequence to P-450dNIR of Fusarium oxysporum and P-450SU2 of Streptomyces griseolus. The recombinant S. lividans strain containing the P-450VD25 gene on a multicopy plasmid converted vitamin D-3 in the medium into 25-hydroxyvitamin D-3 at a maximum yield of 10%.

Vitamin D production after UVB exposure - a comparison of exposed skin regions.

BACKGROUND: Cholecalciferol is an essential steroid produced in the skin by solar ultraviolet B radiation (UVB 290-315nm). Skin production of cholecalciferol depends on factors affecting UVB flux, age and exposed skin area. PURPOSE: Serum cholecalciferol and 25-hydroxyvitamin D3 [25(OH)D3] concentrations were measured after UVB irradiation of 3 different skin areas to compare the skin capacity to produce vitamin D in different anatomic sites in the same individuals. METHOD: Ten voluntary Caucasians (skin photo type II & III, aged 48±12years (±SD)) were exposed to broadband UVB (280-320nm) between February and April. Hands and face, upper body and whole body were exposed to a suberythemic dose of UVB (median 101mJ/cm(2) (min 66, max 143)) (for 3 subsequent days 24h apart with a wash out period of about 3weeks (median 18days (min 11, max 25)) between the exposures of respective area. Serum concentrations of cholecalciferol and 25(OH)D3, were measured immediately before the first and 24h after the last dose of radiation. RESULTS: There was a significantly higher increase in serum cholecalciferol after UVB exposure of the two larger skin areas compared to face and hands, but no difference in increase was found between upper body and whole body exposures. CONCLUSION: Exposure of a larger skin area was superior to small areas and gave greater increase in both serum cholecalciferol and serum 25(OH)D3 concentrations. However, exposure of face and hands, i.e. only 5% of the body surface area, was capable of increasing serum concentrations of 25(OH)D3.

Importancia de la Vitamina D en la época del COVID-19 / Importance of Vitamin D in the time of COVID-19

"Los coronavirus pertenecen a una gran familia de virus (Coronaviridae) que infectan aves y varios mamíferos. El coronavirus actualmente denominado SARS-CoV-2, fue descubierto en diciembre de 2019 en Wuhan, provincia de Hubei, China, y es el agente causal de la epidemia de neumonía atípica actual" (COVID-19; Coronavirus Disease 2019). Los casos más graves presentan un síndrome de dificultad respiratoria aguda que puede conducir a la muerte. La vitamina D (VD), además del efecto bien conocido y positivo sobre la salud ósea y la homeostasis del calcio, tiene efecto pleiotrópico en varios órganos, con distribución casi universal del receptor de VD y de las enzimas de metabolización de 25 hidroxivitamina D (25OHD) en las células del organismo. Estas acciones extraesqueléticas dependen de la síntesis en dichas células del metabolito activo 1,25 dihidroxivitamina D por regulación paracrina y autocrina, dependiente de niveles circulantes óptimos de 25OHD. Por sus acciones inmunomoduladora, antiinflamatoria, antimicrobiana, reguladora del sistema renina-angiotensina-aldosterona, favorecedora de la indemnidad del epitelio respiratorio y la homeostasis redox celular, la VD podría tener efecto protector en la infección por COVID-19. Entre los grupos de riesgo para COVID-19 figuran los adultos mayores, obesos, diabéticos, hipertensos, con afecciones cardiovasculares, patologías con mayor incidencia en individuos con hipovitaminosis VD. La suplementación con VD, para alcanzar niveles óptimos de 25OHD de 40-60 ng/ml, podría reducir la incidencia, severidad y riesgo de muerte en la actual pandemia por COVID-19, como medida complementaria mientras se desarrollan la vacuna y otras medicaciones específicas. (AU)

Coronaviruses belong to a large family of viruses (Coronaviridae) that infect birds and various mammals. The novel coronavirus currently known as SARS-CoV-2 was discovered in December 2019 in Wuhan, Hubei province, China and is the causal agent of the current atypical pneumonia epidemic (COVID-19: Coronavirus Disease 2019); The most severe cases present with acute respiratory distress syndrome that can lead to death. Vitamin D (VD) has a pleiotropic effect on several organs, in addition to its wellknown and positive effect on bone health and calcium homeostasis, with an almost universal distribution of the VD receptor and the metabolites of 25hydroxyvitamin D (25OHD) in all cells of the body. These extra-skeletal actions depend on the synthesis of the active metabolite 1,25dihydroxyvitamin D in the cells depending on the optimal circulating levels of 25OHD and though paracrine and autocrine regulation. Due to its immunomodulatory, anti-inflammatory, antimicrobial, and regulatory actions on the renin angiotensin aldosterone system, which favors the compensation of the respiratory epithelium and cellular redox homeostasis, the VD could have a protective effect on COVID-19 infection. Among the risk groups for COVID-19 are obese, diabetic, and hypertensive patients, subjects with cardiovascular conditions, and elderly people. All these pathologies show a higher incidence in individuals with VD hypovitaminosis. VD supplementation, to achieve optimal 25OHD levels of 40-60 ng/ml, could reduce the incidence, severity, and risk of death in the current COVID-19 pandemic, as a complementary measure while the vaccine and other specific therapies are being developed. (AU)

The involvement of intracellular calcium ion concentration and calmodulin in the 25-hydroxylation of cholecalciferol in ovine and rat liver.

The effect of Ca2+ ion concentration on the 25 hydroxylation of tritiated cholecalciferol (3HD3) was investigated using homogenates of ovine liver from vitamin D replete sheep. A significant decrease in the production of 25 hydroxycholecalciferol (25OHD3) was observed when the concentration of Ca2+ in the homogenate was raised above 0.68 mmol/l by the addition of calcium gluconate. Similarly, a final concentration of 37 mumol EGTA/1 (equivalent to a Ca2+ concentration of 26.5 nmol/l) was associated with a 50% reduction of 25OHD3 production. That is, a broad bell-shaped relationship was observed between the production of 25OHD3 and the Ca2+ concentration in the homogenate. These changes in the rate of production of 25OHD3 were reproduced with hepatocytes from vitamin D replete rats, prepared by collagenase perfusion, using the drugs dantrolene sodium (DaNa) to reduce (ED50 = 57 mmol/l) and veratridine to increase (ED50 = 550 mmol/l) the intracellular Ca2+ concentration. Hepatocytes from vitamin D replete rats also showed a reduction in 25 hydroxylation of D3 (ED50 = 6 ng/ml) in response to the addition of 1-25 dihydroxycholecalciferol (1-25 (OH)2D3). The calmodulin antagonists; W7, compound 48/80, trifluoperazine (TFP) and calmidazolium (R24571) were all found to effect a dose response inhibition of the 25 hydroxylation of cholecalciferol by homogenates of ovine liver. R24571 had a similar inhibitory effect (ED50 = 70 mumol/l) upon the 25 hydroxylase enzyme of rat hepatocytes. It is concluded that the 25 hydroxylation of cholecalciferol in liver of vitamin D replete rats and sheep is calcium sensitive and is reduced in the presence of increased concentrations of 1,25(OH)2D3. Calmodulin may also be involved in the regulation of hepatocyte 25-hydroxylase activity by Ca2+.