Impaired local Vitamin D3 metabolism contributes to IL-36g overproduction in epithelial cells in chronic rhinosinusitis with nasal polyps.

BACKGROUND: Vitamin D (VD) possesses immunomodulatory properties, but its role in chronic rhinosinusitis with nasal polyps (CRSwNP) remains poorly studied. Herein, we aim to explore the regulation and function of VD3 in CRSwNP. METHODS: 25-hydroxyvitamin D3 (25VD3) levels in serum and tissue lysates were detected by ELISA. The expression of VD receptor (VDR) and cytochrome P450 family 27 subfamily B member 1 (CYP27B1), the enzyme that converts 25VD3 to the active 1,25-hydroxyvitamin D3 (1,25VD3), and their expression regulation in human nasal epithelial cells (HNECs) were studied by RT-PCR, western blotting, immunofluorescence, and flow cytometry. RNA sequencing was performed to identify genes regulated by 1,25VD3 in HNECs. HNECs and polyp tissue explants were treated with 1,25VD3, 25VD3, and dexamethasone. RESULTS: 25VD3 levels in serum and nasal tissue lysates were decreased in patients with eosinophilic and noneosinophilic CRSwNP than control subjects. The expression of VDR and CYP27B1 were reduced in eosinophilic and noneosinophilic CRSwNP, particularly in nasal epithelial cells. VDR and CYP27B1 expression in HNECs were downregulated by interferon y and poly (I:C). Polyp-derived epithelial cells demonstrated an impaired ability to convert 25VD3 to 1,25VD3 than control tissues. 1,25VD3 and 25VD3 suppressed IL-36y production in HNECs and polyp tissues, and the effect of 25VD3 was abolished by siCYP27B1 treatment. Tissue 25VD3 levels negatively correlated with IL-36y expression and neutrophilic inflammation in CRSwNP. CONCLUSION: Reduced systemic 25VD3 level, local 1,25VD3 generation and VDR expression result in impaired VD3 signaling activation in nasal epithelial cells, thereby exaggerating IL-36y production and neutrophilic inflammation in CRSwNP.

25(OH)D3 improves granulosa cell proliferation and IVF pregnancy outcomes in patients with endometriosis by increasing G2M+S phase cells.

BACKGROUND: The 25-hydroxyvitamin D3 (25 (OH) D3) is crucial for follicular development. This study aimed to investigate the relationship between the level of 25 (OH) D3 in endometriosis patients, pregnancy outcomes of in vitro fertilization (IVF), and the underlying mechanism. METHODS: The 25 (OH) D3 levels in serum and follicular Fluid (FF) samples were detected using enzyme-linked immunosorbent assay (ELISA). Clinical features and pregnancy outcomes of endometriosis patients were also compared between the deficient group (< 20 ug/ml) and the adequate group (≥ 20 ug/ml). The effects of 25 (OH) D3 on the proliferation and cell cycle of human ovarian granulosa cells were respectively detected by CCK-8 assay and flow cytometry (FCM). The differentially expressed genes (DEGs) in granulosa cells of endometriosis and tubal infertility patients were screened from GEO database. The effects of 25 (OH) D3 on the expressions of CDKN2D, PPARA, TGFB2 and THBD were determined using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot. RESULTS: The levels of 25 (OH) D3 in serum and FF samples were decreased in endometriosis patients. The deficient group had fewer embryos that can be transferred, lower quality embryos and lower clinical pregnancy rates. Adequate 25 (OH) D3 levels in FF samples was a protective factor for live birth outcome in endometriosis patients. 25 (OH) D3 enhanced the proliferation capacity of granulosa cells (the concentration of 10 nM was the most significant) and increased the proportion of G2M + S phase cells. The expression of CDKN2D was decreased and TGFB2 and THBD were significantly upregulated. CONCLUSIONS: 25 (OH) D3 deficiency may be associated with poor IVF pregnancy outcomes in endometriosis patients. 25 (OH) D3 promotes ovarian granulosa cell proliferation by promoting the ability of cells to divide, and may accelerate cell cycle progression by up-regulating THBD and down-regulating CDKN2D expression.

Changes in miRNA expression in the lungs of pigs supplemented with different levels and forms of vitamin D.

BACKGROUND: Vitamin D is an immunomodulator, and its effects have been linked to many diseases, including the pathogenesis of cancer. However, the effect of vitamin D supplementation on the regulation of gene expression of the lungs is not fully understood. This study aims to determine the effect of the increased dose of cholecalciferol and a combination of cholecalciferol + calcidiol, as well as the replacement of cholecalciferol with calcidiol, on the miRNA profile of healthy swine lungs. METHODS AND RESULTS: The swine were long-term (88 days) supplemented with a standard dose (2000IU/kg) of cholecalciferol and calcidiol, the increased dose (3000 IU/kg) of cholecalciferol, and the cholecalciferol + calcidiol combination: grower: 3000 IU/Kg of vitamin D (67% of cholecalciferol and 33% of calcidiol), finisher 2500 IU/Kg of vitamin D (60% of cholecalciferol and 40% of calcidiol). Swine lung tissue was used for Next Generation Sequencing (NGS) of miRNA. Long-term supplementation with the cholecalciferol + calcidiol combination caused significant changes in the miRNA profile. They embraced altered levels of the expression of miR-150, miR-193, miR-145, miR-574, miR-340, miR-381, miR-148 and miR-96 (q-value < 0.05). In contrast, raising the dose of cholecalciferol only changed the expression of miR-215, and the total replacement of cholecalciferol with calcidiol did not significantly affect the miRNAome profile. CONCLUSIONS: The functional analysis of differentially expressed miRNAs suggests that the use of the increased dose of the cholecalciferol + calcidiol combination may affect tumorigenesis processes through, inter alia, modulation of gene regulation of the TGF- ß pathway and pathways related to metabolism and synthesis of glycan.

Effect of source and amount of vitamin D on serum concentrations and retention of calcium, magnesium, and phosphorus in dairy cows.

The objectives of the experiment were to determine the effects of supplementing 2 amounts of 25-hydroxyvitamin D3 (calcidiol; CAL) compared with equal amounts of vitamin D3 (cholecalciferol; CHOL) on serum concentrations, absorptions, and retentions of Ca, Mg, and P in periparturient dairy cows. One hundred seventy-seven (133 parous and 44 nulliparous) pregnant Holstein cows were enrolled in the experiment. Cows were blocked by parity and previous lactation milk yield (parous) or genetic merit for energy-corrected milk yield (nulliparous) and assigned randomly to receive 1 or 3 mg/d of CAL or CHOL in a 2 × 2 factorial arrangement of treatments. Treatments were provided to individual cows as a top-dress to the prepartum diet from 250 d gestation until parturition. The prepartum diet had a dietary cation-anion difference of -128 mEq/kg of dry matter. All cows were fed a common postpartum diet containing 46 µg of vitamin D3/kg of dry matter without further supplementation of treatments. Concentrations of vitamin D metabolites, Ca, Mg, and P in serum were measured pre- and postpartum, in addition to total-tract digestibility and urinary excretion of Ca, Mg, and P in the prepartum period. Feeding 3 mg compared with 1 mg of CAL increased serum 25-hydroxyvitamin D3 (CAL1 = 94 vs. CAL3 = 173 ± 3 ng/mL). In comparison, the increment in serum 25-hydroxyvitamin D3 from feeding 3 mg compared with 1 mg of CHOL was small (CHOL1 = 58 vs. CHOL3 = 64 ± 3 ng/mL). Feeding CAL increased prepartum concentration of P in serum compared with CHOL (CHOL = 1.87 vs. CAL = 2.01 ± 0.02 mM), regardless of the amount fed, but neither source nor amount affected prepartum Ca or Mg in serum. Feeding CAL increased serum Ca and P for the first 11 d postpartum compared with CHOL (CHOL = 2.12 vs. CAL = 2.16 ± 0.01 mM serum Ca; CHOL = 1.70 vs. CAL = 1.78 ± 0.02 mM serum P) but the amount of vitamin D did not affect postpartum concentrations of Ca, Mg, and P in serum. Feeding CAL increased prepartum apparent digestibility of Ca compared with CHOL (CHOL = 26.6 vs. CAL = 33.5 ± 2.8%) but treatments did not affect Ca retention prepartum. Neither source nor amount of vitamin D affected Mg and P apparent digestibility, but CAL decreased the concentration of P excreted in urine during the prepartum period (CHOL = 1.8 vs. CAL = 0.8 ± 0.3 g/d). Calcidiol tended to increase the amount of Ca secreted in colostrum (CHOL = 9.1 vs. CAL = 11.2 ± 0.9 g/d) and Ca excreted in urine postpartum (CHOL = 0.4 vs. CAL = 0.6 ± 0.1 g/d) compared with CHOL. Collectively, feeding CAL at 1 or 3 mg/d compared with CHOL in the last 24 d of gestation is an effective way to increase periparturient serum P concentration and postpartum serum Ca of dairy cows fed a prepartum diet with negative DCAD.

Effect of prepartum source and amount of vitamin D supplementation on lactation performance of dairy cows.

The objectives of this experiment were to determine the effects of supplementing 25-hydroxyvitamin D3 (calcidiol, CAL) compared with vitamin D3 (cholecalciferol, CHOL) at 1 or 3 mg/d in late gestation on production outcomes of dairy cows. One hundred thirty-three parous and 44 nulliparous pregnant Holstein cows were enrolled in the experiment. Cows were blocked by parity and previous lactation milk yield (parous) or genetic merit (nulliparous) and assigned randomly to receive 1 or 3 mg/d of CAL or CHOL in a 2 × 2 factorial arrangement of treatments (CAL1, CAL3, CHOL1, and CHOL3). Treatments were provided to individual cows as a top-dress to the prepartum diet from 250 d in gestation until parturition. The prepartum diet had a dietary cation-anion difference of -128 mEq/kg of dry matter. Production and disease were evaluated for the first 42 d in milk, and reproduction was evaluated to 300 d in milk. Incidence of postpartum diseases did not differ among treatments. Feeding CAL compared with CHOL increased yields of colostrum and colostrum fat, protein, and total solids, resulting in an increased amount of net energy for lactation secreted as colostrum (CHOL = 7.0 vs. CAL = 9.0 ± 0.7 Mcal). An interaction between source and amount was observed for milk yield: CAL3 increased milk yield compared with CHOL3 (CHOL3 = 34.1 vs. CAL3 = 38.7 ± 1.4 kg/d) but milk yield did not differ between CAL1 and CHOL1 (CHOL1 = 36.9 vs. CAL1 = 36.4 ± 1.4 kg/d). Concentrations of serum calcidiol on day of calving and average serum Ca from d 2 to 11 postpartum were positively associated with milk yield in the first 42 d in milk. Interactions between source and amount of vitamin D were also observed for pregnancy after first AI: the percentage of cows receiving CHOL1 and CAL3 that became pregnant was smaller than that of cows receiving CHOL3 and CAL1. However, pregnancy per AI and pregnancy by 300 d in milk did not differ among treatments. Overall, CAL3 increased milk yield compared with CHOL3, whereas in cows fed 1 mg/d (CAL1 and CHOL1), the source of vitamin D generally had no effect. The effect of CAL3 may be explained in part by serum CAL concentrations and postpartum serum Ca, which were associated with milk yield.

Effect of vitamin D metabolites and gene expression of vitamin D receptor, and 1-alpha-hydroxylase related to the sperm quality.

The incidence of male fertility disorders has increased greatly due to various genetic and lifestyle factors. Recently, it has been hypothesized that vitamin D may be involved with idiopathic infertility. The goal of the study was to determine the effect and relationship between blood vitamin D metabolites, intracellular sperm vitamin D levels, and gene expression of 1-α-hydroxylase and VDR, with regard to semen quality. Seventy volunteers aged 25-45 were involved in the study. According to spermogram analysis, participants were stratified into normozoospermic control group, non-normozoospermic target group, and oligoasthenoteratozoospermic group. Vitamin D metabolites (total 25-hydroxycholecalciferol, 1,25-dihydroxycholecalciferol) in blood and spermatozoa were determined by ELISA. Free and bioavailable 25-hydroxycholecalciferol were calculated using the Vermeulen equation. mRNA expression of VDR and 1-α hydroxylase was evaluated by qPCR. Free and bioavailable 25-hydroxycholecalciferol were significantly higher in the control group compared to the target group and compared to the oligoasthenoteratozoospermic group . Intracellular sperm 1,25-dihydroxycholecalciferol was higher in the control group compared to the target group. The mRNA levels of 1- α-hydroxylase were significantly higher in the control samples, while VDR expression was significantly higher in the target group. Significant positive correlations were established between free and bioavailable 25-hydroxycholecalciferol with sperm motility and morphology. Vitamin D metabolites in blood and intracellular sperm 1,25-dihydroxycholecalciferol seem to exert beneficial effects on sperm motility and morphology. Regarding sperm quality, these effects are more pronounced in the free and bioavailable 25OHD compared to the total 25OHD in blood. Higher expression of 1-α-hydroxylase likely leads to higher intracellular levels of 1,25-dihydroxycholecalciferol, which could contribute to sperm motility and morphology. Higher VDR expression may be a compensatory mechanism related to lower intracellular sperm 1,25-dihydroxycholecalciferol.

Elucidation of metabolic pathways of 25-hydroxyvitamin D3 mediated by CYP24A1 and CYP3A using Cyp24a1 knockout rats generated by CRISPR/Cas9 system.

CYP24A1-deficient (Cyp24a1 KO) rats were generated using the CRISPER/Cas9 system to investigate CYP24A1-dependent or -independent metabolism of 25(OH)D3, the prohormone of calcitriol. Plasma 25(OH)D3 concentrations in Cyp24a1 KO rats were approximately twofold higher than in wild-type rats. Wild-type rats showed five metabolites of 25(OH)D3 in plasma following oral administration of 25(OH)D3, and these metabolites were not detected in Cyp24a1 KO rats. Among these metabolites, 25(OH)D3-26,23-lactone was identified as the second major metabolite with a significantly higher Tmax value than others. When 23S,25(OH)2D3 was administered to Cyp24a1 KO rats, neither 23,25,26(OH)3D3 nor 25(OH)D3-26,23-lactone was observed. However, when 23S,25R,26(OH)3D3 was administered to Cyp24a1 KO rats, plasma 25(OH)D3-26,23-lactone was detected. These results suggested that CYP24A1 is responsible for the conversion of 25(OH)D3 to 23,25,26(OH)3D3 via 23,25(OH)2D3, but enzyme(s) other than CYP24A1 may be involved in the conversion of 23,25,26(OH)3D3 to 25(OH)D3-26,23-lactone. Enzymatic studies using recombinant human CYP species and the inhibitory effects of ketoconazole suggested that CYP3A plays an essential role in the conversion of 23,25,26(OH)3D3 into 25(OH)D3-26,23-lactone in both rats and humans. Taken together, our data indicate that Cyp24a1 KO rats are valuable for metabolic studies of vitamin D and its analogs. In addition, long-term administration of 25(OH)D3 to Cyp24a1 KO rats at 110 µg/kg body weight/day resulted in significant weight loss and ectopic calcification. Thus, Cyp24a1 KO rats could represent an important model for studying renal diseases originating from CYP24A1 dysfunction.

Physiological levels of 25-hydroxyvitamin D3 induce a suppressive CD4+ T cell phenotype not reflected in the epigenetic landscape.

1,25-dihydroxyvitamin D3 (1,25(OH)2 D3 ), the active metabolite of vitamin D3 has a strong impact on the differentiation and function of immune cells. Here we analysed the influence of its precursor 25-hydroxyvitamin D3 (25(OH)D3 ) on the differentiation of human CD4+ T cells applying physiological concentrations in vitro. Our data show that 25(OH)D3 is converted to its active form 1,25(OH)2 D3 by T cells, which in turn supports FOXP3, CD25 and CTLA-4 expression and inhibits IFN-γ production. These changes were not reflected in the demethylation of the respective promoters. Furthermore, we investigated the impact of vitamin D3 metabolites under induced Treg (iTreg) polarization conditions using TGF-ß. Surprisingly, no additive effect but a decreased percentage of FOXP3 expressing cells was observed. However, the combination of 25(OH)D3 or 1,25(OH)2 D3 together with TGF-ß further upregulated CD25 and CTLA-4 and significantly increased soluble CTLA-4 and IL-10 secretion whereas IFN-γ expression of iTreg was decreased. Our data suggest that physiological levels of 25(OH)D3 act as potent modulator of human CD4+ T cells and autocrine or paracrine production of 1,25(OH)2 D3 by T cells might be crucial for the local regulation of an adaptive immune response. However, since no epigenetic changes are detected by 25(OH)D3 a rather transient phenotype is induced.

The First Convergent Synthesis of 23,23-Difluoro-25-hydroxyvitamin D3 and Its 24-Hydroxy Derivatives: Preliminary Assessment of Biological Activities.

In this paper, we report an efficient synthetic route for the 23,23-difluoro-25-hydroxyvitamin D3 (5) and its 24-hydroxylated analogues (7,8), which are candidates for the CYP24A1 main metabolites of 5. The key fragments, 23,23-difluoro-CD-ring precursors (9-11), were synthesized starting from Inhoffen-Lythgoe diol (12), and introduction of the C23 difluoro unit to α-ketoester (19) was achieved using N,N-diethylaminosulfur trifluoride (DAST). Preliminary biological evaluation revealed that 23,23-F2-25(OH)D3 (5) showed approximately eight times higher resistance to CYP24A1 metabolism and 12 times lower VDR-binding affinity than its nonfluorinated counterpart 25(OH)D3 (1).

Effects of phytase and 25-hydroxyvitamin D3 supplementation on growth performance and bone development in weaned piglets in Ca- and P-deficient dietary.

BACKGROUND: The beneficial function of phytase and 25-hydroxyvitamin D3 (HyD) on the feed utilization rate has been widely investigated. However, studies concerning its influence on weaned piglets largely lag behind. The aim of this study was to investigate the effects of phytase and HyD supplementation on the growth performance and bone development in weaned piglets under dietary Ca and P deficiency. RESULTS: The results showed that dietary Ca and P deficiency decreased (P < 0.05) the content of serum P in 6-10 kg piglets, as well as reducing (P < 0.05) the contents of serum Ca and P, average daily gain (ADG), bone mineral density (BMD), breaking force (BF), bone ash and femur Ca in 10-20 kg piglets. Compared with the control group, the feed-to-gain ratio (F/G) of 6-10 kg piglets in the Phy group was decreased (P < 0.05), whereas the ADG, blood Ca and P, BMD, BF, bone ash, P apparent digestibility, Ca and P retention rate of 10-20 kg piglets were increased (P < 0.05). The contents of serum osteocalcin and HyD in 6-10 kg piglets and ADG were higher than in the control group (P < 0.05), as well as the contents of serum Ca and HyD in 10-20 kg piglets in the HyD treatment group. Supplementation with both Phy and HyD decreased the F/D (P < 0.05) and increased the contents of serum Ca, P and HyD in 6-10 kg piglets as well as enhancing the ADG, BMD, BF, bone ash, femur Ca and P, serum Ca and P, HyD, and the apparent digestibility and retention of Ca and P (P < 0.05) in 10-20 kg piglets. Supplementation with Phy and HyD in Ca- and P-deficient dietary decreased bone resorption, and improved tight arrangement of collagen fibers and oblique fibers in weaned piglets. CONCLUSION: These data indicated that supplementation with both 1500 U kg-1 Phy and 50 µg kg-1 HyD could enhance dietary Ca and P utilization and promote bone development in low Ca and P dietary, and supplementation with both Phy and HyD had a significant synergy effect compared to single supplement. © 2021 Society of Chemical Industry.

Effects of 25-hydroxyvitamin D3 on growth performance, serum parameters, fecal microbiota, and metabolites in weaned piglets fed diets with low calcium and phosphorus.

BACKGROUND: With the development of intensive farming, long-term exposure of pigs to poor light conditions is not conducive to the production of vitamin D3 , and vitamin D3 deficiency could affect absorption and metabolism of calcium (Ca) and phosphorus (P). 25-Hydroxyvitamin D3 (25OHD3 ) has higher bioactivity than regular vitamin D3 . This study investigated the effects of 25OHD3 on performance, serum parameters, fecal microbiota, and metabolites in weaned piglets fed with low Ca-P diet. RESULTS: It was found that a low Ca-P diet supplemented with 50 µg/kg 25OHD3 (NC + 25-D) improved (P < 0.05) average daily gain (ADG) in phase 2 and in the overall period of the experiment, and increased (P < 0.05) the immunoglobulin G (IgG), immunoglobulin A (IgA), catalase (CAT), bone-specific alkaline phosphatase (BALP), and osteocalcin (OC) serum content on day 28 compared with a low Ca-P diet (NC), but no differences were observed between a normal Ca-P diet (PC) and the NC + 25-D diet. Compared with NC, the abundance of Firmicutes was higher (P < 0.05) in PC and NC + 25-D. NC + 25-D decreased (P < 0.05) the abundance of Streptococcaceae compared with PC and NC, and increased (P < 0.05) the abundance of Lachnospiraceae compared with NC. Serum 25OHD3 was negatively correlated with the abundance of fecal Streptococcaceae (P < 0.05), and positively correlated with the abundance of fecal Lachnospiraceae (P < 0.05). CONCLUSION: Supplementation of 25OHD3 in a low Ca-P diet improved serum immunity, bone biochemical parameters, and fecal microbiota such as decreased Streptococcaceae abundance and increased Lachnospiraceae abundance, which could subsequently promote growth of piglets. The effects were similar to that of a normal Ca-P diet. © 2021 Society of Chemical Industry.

A Fluidic Isolation-Assisted Homogeneous-Flow-Pressure Chip-Solid Phase Extraction-Mass Spectrometry System for Online Dynamic Monitoring of 25-Hydroxyvitamin D3 Biotransformation in Cells.

It is well known that cell can response to various chemical and mechanical stimuli. Therefore, flow pressure variation induced by sample loading and elution should be small enough to ignore the physical impact on cells when we use a Chip-SPE-MS system for cells. However, most existent Chip-SPE-MS systems ignored the pressure alternation because it is extremely difficult to develop a homogeneous-flow-pressure hyphenated module. Herein, we developed an interesting fluidic isolation-assisted homogeneous-flow-pressure Chip-SPE-MS system and demonstrated that it is adequate for online high-throughput determination and quantification of the 25-hydroxyvitamin D3 (25(OH)D3) biotransformation in different cells. Briefly, the homogeneous ambient flow pressure is achieved by fluidic isolation between the cell culture channel and the SPE column, and an automatic sampling probe could accomplish the sample loading and dispensing to fulfill online pretreatment of the sample. Through this new system, the expression levels of 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) can be determined in real time with a detection limit of 2.54 nM. In addition, the results revealed that 25(OH)D3 metabolic activity differed significantly between normal L-02 cells and cancerous HepG2 cells. Treatment of L-02 cells with a high dose of 25(OH)D3 was found to increase significant formation of 24,25(OH)2D3, but this change was not apparent in HepG2 cells. The presented system promises to be a versatile tool for online accurate molecule biotransformation investigation and drug screening processes.

Vitamin D Metabolism Is Dysregulated in Asthma and Chronic Obstructive Pulmonary Disease.

Rationale: Vitamin D deficiency is common in patients with asthma and chronic obstructive pulmonary disease (COPD). Low 25-hydroxyvitamin D (25[OH]D) levels may represent a cause or a consequence of these conditions.Objectives: To determine whether vitamin D metabolism is altered in asthma or COPD.Methods: We conducted a longitudinal study in 186 adults to determine whether the 25(OH)D response to six oral doses of 3 mg vitamin D3, administered over 1 year, differed between those with asthma or COPD versus control subjects. Serum concentrations of vitamin D3, 25(OH)D3, and 1α,25-dihydroxyvitamin D3 (1α,25[OH]2D3) were determined presupplementation and postsupplementation in 93 adults with asthma, COPD, or neither condition, and metabolite-to-parent compound molar ratios were compared between groups to estimate hydroxylase activity. Additionally, we analyzed 14 datasets to compare expression of 1α,25(OH)2D3-inducible gene expression signatures in clinical samples taken from adults with asthma or COPD versus control subjects.Measurements and Main Results: The mean postsupplementation 25(OH)D increase in participants with asthma (20.9 nmol/L) and COPD (21.5 nmol/L) was lower than in control subjects (39.8 nmol/L; P = 0.001). Compared with control subjects, patients with asthma and COPD had lower molar ratios of 25(OH)D3-to-vitamin D3 and higher molar ratios of 1α,25(OH)2D3-to-25(OH)D3 both presupplementation and postsupplementation (P ≤ 0.005). Intergroup differences in 1α,25(OH)2D3-inducible gene expression signatures were modest and variable if statistically significant.Conclusions: Attenuation of the 25(OH)D response to vitamin D supplementation in asthma and COPD associated with reduced molar ratios of 25(OH)D3-to-vitamin D3 and increased molar ratios of 1α,25(OH)2D3-to-25(OH)D3 in serum, suggesting that vitamin D metabolism is dysregulated in these conditions.

A novel caged Cookson-type reagent toward a practical vitamin D derivatization method for mass spectrometric analyses.

RATIONALE: 25-Hydroxylated vitamin D is the best marker for vitamin D (VD). Due to its low ionization efficiency, a Cookson-type reagent, 1,2,4-triazoline-3,5-dione (TAD), is used to improve the detection/quantification of VD metabolites by liquid chromatography/tandem mass spectrometry (LC/MS/MS). However, the high reactivity of TAD makes its solution stability low and inconvenient for practical use. We here describe the development of a novel caged Cookson-type reagent, and we assess its performances in the quantitative and differential detection of four VD metabolites in serum using LC/MS/MS. METHODS: Caged 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) analogues were prepared from 4-(4'-dimethylaminophenyl)-1,2,4-triazolidine-3,5-dione. Their stability and reactivity were examined. The optimized caged DAPTAD (14-(4-(dimethylamino)phenyl)-9-phenyl-9,10-dihydro-9,10-[1,2]epitriazoloanthracene-13,15-dione, DAP-PA) was used for LC/MS/MS analyses of VD metabolites. RESULTS: The solution stability of DAP-PA in ethyl acetate dramatically improved compared with that of the non-caged one. We measured the thermal retro-Diels-Alder reaction enabling the release of DAPTAD and found that the derivatization reaction was temperature-dependent. We also determined the detection limit and the lower limit of quantifications for four VD metabolites with DAPTAD derivatization. CONCLUSIONS: DAP-PA was stable enough for mid- to long-term storage in solution. This advantage shall contribute to the detection and quantification of VD in clinical laboratories, and as such to the broader use of clinical mass spectrometry.

25(OH) D3 alleviate liver NK cytotoxicity in acute but not in chronic fibrosis model of BALB/c mice due to modulations in vitamin D receptor.

BACKGROUND: Low 25-Hydroxy-vitamin-D; "25(OH)-D3" serum and vitamin D receptor (VDR) levels were recently correlated to advanced fibrosis. However, VDR mechanism in liver fibrosis modulations is not well understood. In this study, we aimed to evaluate changes in liver NK cells cytotoxicity due to modulations in VDR in CCl4 fibrosis model following 25(OH) D3 injections. METHODS: Carbon-tetrachloride (CCl4) hepatic-fibrosis was induced in BALB/c mice for 1 and 4 weeks as an acute and chronic fibrosis model, respectively. Along 1th to 4th weeks, vitamin D were i.p injected/2x week. Liver were assessed histologically and for proteins quantification for VDR and αSMA expressions. In vitro, potential killing of NK cells were evaluated following co-culture with primary-hepatic-stellate-cells (pHSCs) obtained from BALB/c WT-mice. RESULTS: Systemic inflammation and hepatic-fibrosis increased along 4 weeks of CCl4 as indicated by serum ALT and αSMA expressions (P < 0.02) as well as histological assessments, respectively. These results were associated with increased NK1.1 activations and hypercalcemia. While vitamin D administrations delayed fibrosis of early stages, vitamin D worsen hepatic-fibrosis of late stages of CCl4. In week 4, no further activations of NK cells were seen following vitamin D injections and were associated with down-expressions of VDR (1.7 Fold, P < 0.004) indicating the inability of vitamin D to ameliorate hepatic fibrosis. In vitro, NK cells from the chronic model of CCl4 did not affect pHSCs killing and fail to reduce fibrosis. CONCLUSION: Vitamin D alleviate liver NK cytotoxicity in acute but not in chronic fibrosis model due to modulations in vitamin D receptor and calcium. Hypercalcemia associated with late fibrosis may inhibited VDR levels, however, may not explain the profibrogenic effects of vitamin D.

Efficient biotransformation of vitamin D3 to 25-hydroxyvitamin D3 by a newly isolated Bacillus cereus strain.

25-hydroxyvitamin D3 has attracted considerable attention due to its great medical value and huge market demand in animal husbandry. Microbial production of 25-hydroxyvitamin D3 has been recognized as an alternative superior to traditional chemical synthesis. In this study, a Gram-positive bacteria zju 4-2 (CCTCC M 2019385) was isolated from the soil using vitamin D3 as the sole carbon source and was identified as Bacillus cereus according to its physiological characteristics and 16S rRNA analysis, which also showed a relatively high capacity for 25-hydroxyvitamin D3 production. Through systematic optimization of different catalytic conditions, the optimal solvent system of vitamin D3, vitamin D3 addition time and concentration, temperature, and pH were shown to be propylene glycol/ethanol (v/v = 9:1), early stationary phase, 2 g/L, 37 °C, and pH 7.2, respectively. With these optimal conditions, 796 mg/L of 25-hydroxyvitamin D3 was achieved after 48 h bioconversion with zju 4-2 at the shake flask level. Finally, up to 830 mg/L 25-hydroxyvitamin D3 with a yield of 41.5% was obtained in a 5 L fermentation tank. Our developed biotransformation process with this newly isolated strain provides a platform to produce 25-hydroxyvitamin D3 efficiently at industrialization scale.

Effects of maternal 25-hydroxycholecalciferol during the last week of gestation and lactation on serum parameters, intestinal morphology and microbiota in suckling piglets.

This study was conducted to test the effects of maternal 25-hydroxycholecalciferol (25OHD3) supplementation on serum parameters, intestinal morphology and microbiota in suckling piglets. The experiment started on day 107 of gestation and lasted until piglets were weaned on day 21 of lactation. Thirty-two sows were allocated randomly to two treatments (ND diet, basal diet with 2000 IU/kg of vitamin D3; 25-D diet, basal diet with 50 µg/kg 25OHD3). Results showed that maternal 25-D treatment increased (p < 0.05) serum 25OHD3 concentration in the umbilical cords, which led to higher (p < 0.05) serum 25OHD3 concentration of suckling piglets from 25-D sows. The GSH-Px activity in colostrum was higher (p < 0.05), as well as SOD and GSH-Px activities in milk, were higher (p < 0.05) in 25-D sows than ND sows. Compared with piglets suckling ND sows, piglets suckling 25-D sows had higher (p < 0.05) serum SOD activity on day 7, 14 and 21 of lactation. On day 21 of lactation, piglets form 25-D sows had greater (p < 0.05) serum levels of GH and IGF-I and lower (p < 0.05) serum DAO activity than those from ND sows. Piglets from 25-D sows had higher (p < 0.05) jejunal villus height than those from ND sows. Feeding 25OHD3 to sows tended to increase (p < 0.10) the species richness in the colonic digesta of suckling piglets, as reflected by the α-diversity index of Chao-1. In the caecal digesta, the α-diversity for bacterial community analysis of Simpson and Shannon was lower (p < 0.05) in 25-D piglets than ND piglets. The relative abundances of colonic Alloprevotella and caecal Lactobacillus were significantly higher, while the population of caecal [Eubacterium]_coprostanoligenes_group was lower (p < 0.05) in 25-D piglets than ND piglets. In conclusion, maternal 25OHD3 supplementation partly improved antioxidant status in sows and suckling piglets and altered gut microbiota in the hindgut of piglets.

Catalytic properties of 25-hydroxyvitamin D3 3-epimerase in rat and human liver microsomes.

25-Hydroxyvitamin D3 3-epimerase catalyzes the 3ß â†’ 3α epimerization of 25-hydroxyvitamin D3 (25(OH)D3) producing 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3). 3-Epi-25(OH)D3 is one of the most abundant forms of vitamin D present in the serum. It can be converted to 3-epi-1α,25-dihydroxyvitamin D3 by CYP27B1 which generally displays lower biological activity than 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). The 25(OH)D3 3-epimerase has been poorly characterized to date and the gene encoding it has not been identified. The 3-epimerase has been reported to be present in the microsomal fraction of cells, including liver cells, and to use NADPH as cofactor. It can also act on 1,25(OH)2D3 and 24,25(OH)2D3 forming the 3α-epimers. In this study we have characterized the activity of the 25(OH)D3 3-epimerase in rat and human liver microsomes, using 25(OH)D3 as substrate and HPLC to analyze product formation. For both rat and human liver microsomes the preferred cofactor was NADH, with the rat enzyme displaying a 6-fold greater catalytic efficiency (Vmax/Km) for NADH over that for NADPH. No activity was observed with oxidized cofactor, either NAD+ or NADP+. This was unexpected since the initial step in the epimerization, predicted to be the oxidation of the 3ß-OH to a ketone, would require oxidized cofactor. The rat 3-epimerase in microsomes gave a Km for 25(OH)D3 of 14 µM. The reverse reaction, conversion of 3-epi-25(OH)D3 to 25(OH)D3, was catalyzed by both rat and human liver microsomes but at lower rates than the forward reaction. In conclusion, both rat and human 25-hydroxyvitamin D3 3-epimerase catalyze the reversible interconversion of 25(OH)D3 and 3-epi-25(OH)D3, and use NADH as the preferred cofactor. The lack of requirement for exogenous NAD+ suggests that the enzyme has a tightly bound NAD+ in its active site that is released only upon its reduction.

Optimal sunscreen use, during a sun holiday with a very high ultraviolet index, allows vitamin D synthesis without sunburn.

BACKGROUND: Sunlight contains ultraviolet (UV)A and UVB radiation. UVB is essential for vitamin D synthesis but is the main cause of sunburn and skin cancer. Sunscreen use is advocated to reduce the sun's adverse effects but may compromise vitamin D status. OBJECTIVES: To assess the ability of two intervention sunscreens to inhibit vitamin D synthesis during a week-long sun holiday. METHODS: The impact of sunscreens on vitamin D status was studied during a 1-week sun holiday in Tenerife (28° N). Comparisons were made between two formulations, each with a sun protection factor (SPF) of 15. The UVA-protection factor (PF) was low in one case and high in the other. Healthy Polish volunteers (n = 20 per group) were given the sunscreens and advised on the correct application. Comparisons were also made with discretionary sunscreen use (n = 22) and nonholiday groups (51·8° N, n = 17). Sunscreen use in the intervention groups was measured. Behaviour, UV radiation exposure, clothing cover and sunburn were monitored. Serum 25-hydroxyvitamin D3 [25(OH)D3 ] was assessed by high-performance liquid chromatography-tandem mass spectrometry. RESULTS: Use of intervention sunscreens was the same (P = 0·60), and both equally inhibited sunburn, which was present in the discretionary use group. There was an increase (P < 0·001) in mean ± SD 25(OH)D3 (28·0 ± 16·5 nmol L-1 ) in the discretionary use group. The high and low UVA-PF sunscreen groups showed statistically significant increases (P < 0·001) of 19·0 ± 14·2 and 13·0 ± 11·4 nmol L-1 25(OH)D3 , respectively with P = 0·022 for difference between the intervention sunscreens. The nonholiday group showed a fall (P = 0·08) of 2·5 ± 5·6 nmol L-1 25(OH)D3 . CONCLUSIONS: Sunscreens may be used to prevent sunburn yet allow vitamin D synthesis. A high UVA-PF sunscreen enables significantly higher vitamin D synthesis than a low UVA-PF sunscreen because the former, by default, transmits more UVB than the latter. What's already known about this topic? Action spectra (wavelength dependence) for erythema and the cutaneous formation of vitamin D overlap considerably in the ultraviolet (UV)B region. Theoretically, sunscreens that inhibit erythema should also inhibit vitamin D synthesis. To date, studies on the inhibitory effects of sunscreens on vitamin D synthesis have given conflicting results, possibly, in part, because people typically apply sunscreen suboptimally. Many studies have design flaws. What does this study add? Sunscreens (sun protection factor, SPF 15) applied at sufficient thickness to inhibit sunburn during a week-long holiday with a very high UV index still allow a highly significant improvement of serum 25-hydroxyvitamin D3 concentration. An SPF 15 formulation with high UVA protection enables better vitamin D synthesis than a low UVA protection product. The former allows more UVB transmission.

The possible antidiabetic effects of vitamin D receptors agonist in rat model of type 2 diabetes.

Vitamin D3 deficiency was found to be tightly linked to many health problems including metabolic syndrome, cancer, cardiovascular diseases, and type 2 diabetes mellitus. In our study, we tested the possible antidiabetic effects of one of vitamin D3 analogs, alfacalcidol, solely or in a combination with metformin on type 2 diabetic rats. Type 2 diabetic model rats were induced by feeding high-fat diet for 4 weeks followed by intraperitoneal injection of streptozotocin. In addition to the control group, the diabetic rats were divided into four groups: untreated, metformin-treated, alfacalcidol-treated, and combination-treated group (metformin + alfacalcidol) for 4 weeks. The level of fasting blood glucose, fasting serum insulin, homeostatic model of insulin resistance, serum lipid profile, liver enzymes, calcium, phosphorus, and 25-hydroxyvitamin D3 were also determined. Besides, sterol regulatory element binding protein-1c (SREBP-1c) and vitamin D receptors (VDR) gene expression at mRNA and protein levels were evaluated. The level of significance was fixed at P ≤ 0.05 for all statistical tests. Alfacalcidol, solely or combined with metformin, significantly ameliorated glucose homeostasis and lipid profile parameters (P < 0.001) with a neutral effect on calcium and phosphorus levels. Significant downregulation of mRNA expression of SREBP-1c in the liver, white as well as brown adipose tissues (P < 0.001) and different patterns of mRNA expression of VDR gene in pancreas and white adipose tissue were observed in rats treated with alfacalcidol solely or in combination with metformin. Vitamin D3 analogs can modulate glucose parameters and lipid metabolism in a diabetic rat model and it provides additional protective effects when combined with metformin.