Development of free 25-hydroxyvitamin D3 assay method using liquid chromatography-tandem mass spectrometry.

The free hormone hypothesis has triggered controversies regarding the measurement of free vitamin D metabolites, such as free 25-hydroxyvitamin D (25(OH)D), as a suitable indicator for total vitamin D for clinical use. This issue can be addressed by developing a precise and accurate method for free 25(OH)D measurement. In the present study, a novel assay method for free 25(OH)D3 based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. Sample preparation first involved ultrafiltration to remove vitamin D-binding protein-bound and albumin-bound 25(OH)D, followed by extraction with a column, derivatization, evaporation, dissolution, and injection into the LC-MS/MS system. The coefficient of variation of repeatability and reproducibility obtained were 3.8-4.5% and 4.8-5.9%, respectively. Satisfactory linearity (r=0.999) was obtained up to 80 pg/ml. The lower quantification limit was 0.97 pg/ml and the S/N ratio on the peak of 1.0 pg/ml sample was 24.8 (which is more than the acceptable value of 10). The recovery rate was between 84.5 and 92.4% with a negligible matrix effect (94.5-104.9%). Levels of free 25(OH)D3, but not total 25(OH)D3, in the serum of the patients with chronic kidney disease (CKD) and hepatic cirrhosis (HC) were substantially lower than those in healthy subjects. The correlation coefficient between total and free 25(OH)D3 was 0.738 in all samples, while the linear regression equations were different between the patients with CKD and HC. In conclusion, LC-MS/MS assay for free 25(OH)D3 might be useful to evaluate high-throughput methods, including ELISA.

Graphene oxide and fluorescent aptamer based novel biosensor for detection of 25-hydroxyvitamin D3.

For maintaining the healthy metabolic status, vitamin D is a beneficial metabolite stored majorly in its pre-activated form, 25-hydroxyvitamin D3 (25(OH)D3). Due to its important role in bone strengthening, the study was planned to quantify 25(OH)D3 levels in our blood. Quantification techniques for 25(OH)D3 are costly thus requiring a need for a low cost, and sensitive detection methods. In this work, an economic, and sensitive sensor for the detection of 25(OH)D3 was developed using aptamer and graphene oxide (GO). Aptamer is an oligonucleotide, sensitive towards its target, whereas, GO with 2D nanosheets provides excellent quenching surface. Aptamer labeled with fluorescein (5', 6-FAM) is adsorbed by π-π interaction on the GO sheets leading to quenching of the fluorescence due to Förster resonance energy transfer (FRET). However, in the presence of 25(OH)D3, a major portion of aptamer fluorescence remains unaltered, due to its association with 25(OH)D3. However, in the absence, aptamer fluorescence gets fully quenched. Fluorescence intensity quenching was monitored using fluorescence spectrophotometer and agarose gel based system. The limit of detection of 25(OH)D3 by this method was found to be 0.15 µg/mL whereas when GO-COOH was used, limit of detection was improved to 0.075 µg/mL. Therefore, this method could come up as a new sensing method in the field of vitamin D detection.

Overestimation of 3α- over 3ß-25-Hydroxyvitamin D3 Levels in Serum: A Mechanistic Rationale for the Different Mass Spectral Properties of the Vitamin D Epimers.

The metabolism of vitamin D3 includes a parallel C-3 epimerization pathway-in addition to the standard metabolic processes for vitamin D3-reversing the stereochemical configuration of the -OH group at carbon-3 (ß→α). While the biological function of the 3α epimer has not been elucidated yet, the additional species cannot be neglected in the analytical determination of vitamin D3, as it has the potential to introduce analytical errors if not properly accounted for. Recently, some inconsistent mass spectral behavior was seen for the 25-hydroxyvitamin D3 (25(OH)D3) epimers during quantification using electrospray LC-MS/MS. The present work extends that of Flynn et al. ( Ann. Clin. Biochem. 2014, 51, 352-559) and van den Ouweland et al. ( J. Chromatogr. B 2014, 967, 195-202), who reported larger electrospray ionization response factors for the 3α epimer of 25(OH)D3 in human serum samples as compared to the regular 3ß variant. The present work was concerned with the mechanistic reasons for these differences. We used a combination of electrospray ionization, atmospheric pressure chemical ionization, and density functional theory calculations to uncover structural dissimilarities between the epimers. A plausible mechanism is described based on intramolecular hydrogen bonding in the gas phase, which creates a small difference of proton affinities between the epimers. More importantly, this mechanism allows the explanation of the different ionization efficiencies of the epimers based on kinetic control of the ionization process, where ionization initially takes place at the hydroxyl group with subsequent proton transfer to a basic carbon atom. The barrier for this transfer differs between the epimers and is in direct competition with H2O elimination from the protonated hydroxyl group. The "hidden" site of high gas phase basicity was revealed through computational calculations and appears to be inaccessible via direct protonation.

Candidate reference method for determination of vitamin D from dried blood spot samples.

Background The current millennium has seen an explosion in vitamin D testing with the overarching aim of requests to clinically stratify patients as replete or deficient in vitamin D. At a population level, dried blood spot (DBS) sampling offers a less invasive and more practical application for assessment of vitamin D status. We have therefore aimed to develop a sensitive and robust DBS vitamin D method that is traceable to serum for use in population-based studies. Methods Blood spots, calibrators and controls were prepared by punching a 3.2 mm DBS from filter paper and placed into a 96-well micro-plate. The DBS disk was eluted with a combination of water-methanol and internal standard (ISTD) solution followed by supported-liquid extraction and derivatisation. The extract was analysed by liquid-chromatography tandem-mass spectrometry in positive electrospray-ionisation mode with 732.5 > 673.4 and 738.4 > 679.4 m/z ion-transitions for derivatised vitamin D and the ISTD, respectively. Vitamin D results were made traceable to the National Institute of Standards and Technology reference material through the inclusion of Chromsystems vitamin D calibrators. Results 25-Hydroxy-vitamin D3 and its related ISTD were detected at a retention time of 7 min. The seven-point calibration-curve consistently demonstrated a coefficient of determination of 0.99 with an experimentally determined reportable range of 0.5-376 nmol/L. Method validation studies using DBS samples demonstrated 12.9% between-assay imprecision at 45 nmol/L, 84% average recovery and high correlation with plasma vitamin D (correlation coefficient = 0.86). Conclusions We have successfully developed an analytical method for vitamin D quantitation from DBSs which will be applied to our population-based vitamin D research study. This approach improves traceability of DBS results and potentially could be used broadly for other DBS measurands that require comparison to serum/plasma for their interpretation.

Tyrosine-protein phosphatase non-receptor type 2 inhibits alveolar bone resorption in diabetic periodontitis via dephosphorylating CSF1 receptor.

Tyrosine-protein phosphatase non-receptor type 2 (PTPN2) is an important protection factor for diabetes and periodontitis, but the underlying mechanism remains elusive. This study aimed to identify the substrate of PTPN2 in mediating beneficial effects of 25-Hydroxyvitamin D3 (25(OH)2D3 ) on diabetic periodontitis. 25(OH)2D3 photo-affinity probe was synthesized with the minimalist linker and its efficacy to inhibit alveolar bone loss, and inflammation was evaluated in diabetic periodontitis mice. The probe was used to pull down the lysates of primary gingival fibroblasts. We identified PTPN2 as a direct target of 25(OH)2D3 , which effectively inhibited inflammation and bone resorption in diabetic periodontitis mice. In addition, we found that colony-stimulating factor 1 receptor (CSF1R) rather than JAK/STAT was the substrate of PTPN2 to regulate bone resorption. PTPN2 direct interacted with CSF1R and dephosphorylated Tyr807 residue. In conclusion, PTPN2 dephosphorylates CSF1R at Y807 site and inhibits alveolar bone resorption in diabetic periodontitis mice. PTPN2 and CSF1R are potential targets for the therapy of diabetic periodontitis or other bone loss-related diseases.

Eluted 25-hydroxyvitamin D3 from radially aligned nanofiber scaffolds enhances cathelicidin production while reducing inflammatory response in human immune system-engrafted mice.

Vitamin D3 modulates immune response, induces endogenous antimicrobial peptide production, and enhances innate immunity to defend against infections. These findings suggest that incorporating vitamin D3 into medical devices or scaffolds could positively modulate host immune response and prevent infections. In the current study, we evaluated host responses and endogenous antimicrobial peptide production using 25-hydroxyvitamin D3 (25(OH)D3)-eluting radially aligned PCL nanofiber scaffolds in human immune system-engrafted mice. We transformed traditional 2D electrospun nanofiber membranes into radially aligned PCL nanofiber scaffolds using the concept of solid of revolution and an innovative gas-foaming technique. Such scaffolds can promote rapid cellular infiltration and neovascularization. The infiltrating immune cells within subcutaneously implanted 25(OH)D3-containing scaffolds mainly consisted of human macrophages in the M1 phase (CCR7+), mice macrophages in the M2 phase (CD206+), and human cytotoxic T cells (CD8+) other than few human T-helper cells (CD4+). The 25(OH)D3-eluting nanofiber scaffolds significantly inhibited the production of pro-inflammatory cytokines (TNF-α, IL-6), while accelerating the production of anti-inflammatory cytokines (IL-4, IL-10) within the scaffolds. Additionally, we observed increased expression of human cathelicidin LL-37 within the 25(OH)D3-eluting scaffolds, while no LL-37 expression was observed in the control. Together, these findings support further work in the design of vitamin D3-eluting medical devices or scaffolds for modulating immune response and promoting antimicrobial peptide production. This could potentially reduce the inflammatory response, prevent infections, and eventually improve success rates of implants. STATEMENT OF SIGNIFICANCE: Transplant failure of medical devices, grafts, scaffolds, and tissue-engineered constructs due to inflammation and infection causes not only economic losses but also sufferings of second operation to the patient. Positive modulation of the host response to implants, scaffolds, and tissue-engineered constructs is likely to reduce the failure rate. Vitamin D3 plays an important role in modulating the immune response. It is able to not only reduce inflammation and induce endogenous antimicrobial peptide production but also prevent multidrug resistance and other side effects of traditional antibiotics. In this study, host responses to 25-hydroxyvitamin D3 (25(OH)D3)-eluting radially aligned PCL nanofiber scaffolds were evaluated in human immune system-engrafted mice. The 25(OH)D3-eluting medical devices or scaffolds were able to modulate positive immune response and promote antimicrobial peptide production. This work presented an innate immunity-enhancing approach for reducing the inflammatory response and preventing infections, likely resulting in improvement of success rates of implants.

Development, validation and comparison of four methods for quantifying endogenous 25OH-D3 in human plasma.

To meet the increasing clinical needs for 25-hydroxyvitamin D3 (25OH-D3) detection, the development of an efficient and accurate high-performance liquid chromatography-mass spectrometry (HPLC-MS) method for plasma 25OH-D3 quantitation is important. Since 25OH-D3 is an endogenous compound, the lack of a plasma blank increases the difficulty of accurately quantifying 25OH-D3. Selection of a method suitable for clinical monitoring among various methods for endogenous compound quantification is necessary. Methyl tert butyl ether was chosen for the sample treatment in a liquid-liquid extraction protocol. Water as a blank matrix, 5% human serum albumin in water as a blank matrix, surrogate analyte and background subtraction were designed to address the problem of a deficiency of a plasma blank. Four liquid chromatography-tandem mass spectrometry methods were fully validated to verify the advantages and limitations owing to regulatory deficiencies for endogenous compound validation. All four methods met the criteria and could be used to monitor clinical samples. Overall 30 human plasma samples were quantified in parallel using the four methods. The difference between any two methods was <12.6% and the total relative standard deviation was <5.2%. Background subtraction and 5% human serum albumin in water as a blank matrix may be better choices considering data quality, matrix similarity, cost and practicality.

Vitamin D in Wild and Farmed Atlantic Salmon (Salmo Salar)-What Do We Know?

Salmon have been widely publicized as a good dietary source of vitamin D, but recent data points to large variation in vitamin D content and differences between wild and farmed salmon. We aimed to: (1) investigate the content of vitamin D in Atlantic salmon (Salmo salar) in wild species caught in two different waters, (2) perform a 12-week feeding trial in farmed Salmo salar with 270-1440 µg vitamin D3/kg feed (4-20 times maximum level in the EU) and (3) conduct a review for the published data on the content of vitamin D in salmonids. Content of vitamin D3 in the fillet from wild salmon caught in the Baltic Sea and the North Sea was significantly different (p < 0.05), being 18.5 ± 4.6 µg/100 g and 9.4 ± 1.9 µg/100 g, respectively. In the farmed salmon the content ranged from 2.9 ± 0.7 µg vitamin D3/100 g to 9.5 ± 0.7 µg vitamin D3/100 g. Data from 2018 shows that farmed salmon contained 2.3-7.3 µg vitamin D3/100 g. Information on the content of vitamin D in wild and farmed salmonids is very limited, which calls for further research to ensure a sustainable production of salmon with adequate vitamin D.

Synthesis and characterization of 20-hydroxyvitamin D3 with the A-ring modification.

Two novel 20-hydroxyvitamin D3 analogues (4a,b) with the A-ring modification have been synthesized by a convergent manner. An alternative pathway of vitamin D3 metabolism by cytochrome P450scc CYP11A1 was reported to afford 20-hydroxyvitamin D3 (3), functions of which remain to be explored. Based on the structure of the 20-hydroxy metabolite, novel analogues (4a,b) with the modifications, including the 1α-hydroxy, 25-hydroxy and 2α-methyl groups, have been designed. The side chain of the requisite CD-ring portions (9a,b) was introduced by Grignard reaction as a key step, and the stereochemistry at the C20 position was confirmed by the X-ray crystal structure analysis of the synthetic intermediate (8b). Preliminary biological characterization using the bovine thymus vitamin D receptor suggested that the introduction of the active motifs into the 20-hydroxyvitamin D3 scaffold elevated the receptor affinity.

Measured free 25-hydroxyvitamin D in healthy children and relationship to total 25-hydroxyvitamin D, calculated free 25-hydroxyvitamin D and vitamin D binding protein.

BACKGROUND: vitamin D deficiency in children is still a global health problem. Measuring free 25-hydroxyvitamin D concentrations could provide a better estimate of the vitamin D status than total 25-hydroxyvitamin D (25(OH)D) levels. OBJECTIVE: To assess the relationship between measured free vitamin D (m-f25(OH)D) and calculated free 25(OH)D (c-f25(OH)D), total 25(OH)D, intact parathyroid hormone (iPTH) and other markers of phosphocalcic metabolism. To establish serum m-f25(OH)D concentrations corresponding to a total 25(OH)D > 50 nmol/L which is accepted as vitamin D-sufficiency status in children. DESIGN: Prospective cohort study. SETTING: January and February 2017 in a Mediterranean population. PATIENTS: healthy children. MEASUREMENTS: m-f25(OH)D and vitamin D binding protein (VDBP) by ELISA. Free 25(OH)D was calculated using the formula described by Bikle. RESULTS: m-f25(OH)D directly correlated with total 25(OH)D (r:0.804,p < .001), serum calcium (r:0.26,p:0.035), and c-f25(OH)D (r:0.553,p:0.016); and inversely with iPTH (r:-0.374, p:0.002), alkaline phosphatase (r:-0.28, p:0.026), and age (r:-0.289, p:0.018). Total 25(OH)D correlated with the same parameters as m-f25(OH)D except for serum calcium. However, c-f25(OH)D correlated only with total 25(OH)D and VDBP, both included in the calculation formula. Multiple regression analysis showed that m-f25(OH)D variations were independently explained by calcium (ß:0.156, p:0.026) and total 25(OH)D (ß:0.043, p < .001). The optimal m-f25(OH)D cut-off for discriminating between insufficient and sufficient total 25(OH)D was >9.8 pmol/L (Area Under Curve (AUC): 0.897 (95% confidence interval (CI): (0.798-0.958); p < .001; sensitivity:72.7% (95%CI: 49.8-89.3); specificity: 95.5% (95%CI: 84.5-99.4)). CONCLUSIONS: Directly measured free vitamin D correlated better with markers of phosphocalcic metabolism than total 25(OH)D and c-f25(OH)D in a population of healthy children.

Anti-annexin A5 antibodies and 25-hydroxy-cholecalciferol in female patients with primary antiphospholipid syndrome.

Vascular antiphospholipid syndrome (VAPS) and obstetric (OAPS) are different entities because some patients only develop thrombosis (without recurrent pregnancy losses) and vice versa. Only two articles have reported that low 25-hydroxy-cholecalciferol (vitamin D3, VD3) levels were not correlated with the presence of conventional antiphospholipid antibodies (aPL Abs: anticardiolipin (aCL), anti-beta2glycoprotein I (aß2gpI), and lupus anticoagulant (LA)), but no article analyzed the association of VD3 and anti-annexin A5 (aanxA5) Abs. The aim of our study was to investigate the association between VD3, multiple positivity of conventional aPL and aanxA5 Abs levels only in female OAPS vs. VAPS. Our study included 62 consecutive female PAPS patients. Concentrations of Abs were measured by ELISA, while VD3 levels were determined by immunochemiluminescence. Only 10/62 (16.13%) had sufficient (≥ 30 ng/ml) VD3 levels, while 48/62 (77.42%) and 4/62 (6.45%) had insufficiency and VD3 deficiency, respectively. Statistically significant VD3 deficiency was noticed in VAPS (vs. OAPS, P = 0.013). A negative correlation between VD3 levels and the age of patients was noticed (r = - 0.493, P = 0.032) only in VAPS subgroup. Multiple positivity of aPL and aanxA5 Abs was not associated with VD3 deficiency. Newly emerging aPL Abs, such as aanxA5 Abs, or their combinations with classical aPL Abs are not associated with VD3 deficiency in neither OAPS nor VAPS patients. Due to its immunomodulatory roles in B-Ly homeostasis, supplementation with VD3 should be considered in APS, at least in subgroup with severe form of the disease, i.e., VAPS.

Without 1α-hydroxylation, the gene expression profile of 25(OH)D3 treatment overlaps deeply with that of 1,25(OH)2D3 in prostate cancer cells.

Recently, the antiproliferative action of 1,25(OH)2D3 (1,25D3), an active metabolite of vitamin D3, in the management of prostate cancer has been argued rigorously. In this study, we found that at a physiological concentration, 25(OH)D3 (25D3), the precursor of 1,25D3 and an inactive form of vitamin D because of its much weaker binding activity to the vitamin D receptor (VDR) compared with 1,25D3, had a gene expression profile similar to that of 1,25D3 in prostate cancer LNCaP cells. By immunocytochemistry, western blotting, and CYP27B1 and/or VDR knockdown by small interfering RNAs, we found that 10-7 M 25D3, which is within its uppermost physiological concentration in the bloodstream, induced VDR nuclear import and robustly activated its target genes in the virtual absence of CYP27B1 expression. Comprehensive microarray analyses verified 25D3 bioactivity, and we found that 25D3 target gene profiles largely matched those of 1,25D3, while the presence a small subset of 25D3- or 1,25D3-specific target genes was not excluded. These results indicated that 25D3 shares bioactivity with 1,25D3 without conversion to the latter. Metallothionein 2A was identified as a 1,25D3-specific repressive target gene, which might be a prerequisite for 1,25D3, but not 25D3, to exert its anti-proliferative action in LNCaP cells.

Investigation of 20S-hydroxyvitamin D3 analogs and their 1α-OH derivatives as potent vitamin D receptor agonists with anti-inflammatory activities.

20S-hydroxyvitamin D3 [20S(OH)D3] is anti-inflammatory and not hypercalcemic, suggesting its potential as a lead compound. In this study, side chain modified 20S(OH)D3 analogs (4, 13, 23 and 33) together with their 1α-OH derivatives were synthesized and their metabolism and biological activities tested. 4, 13 and 23 are good substrates for CYP27B1, enabling enzymatic synthesis of their 1α-OH derivatives 5, 14 and 24. However, 33 could not be hydroxylated by CYP27B1 and acts as an inhibitor. All analogs were poorer substrates for CYP24A1 than calcitriol, indicating improved catabolic stability. While the parent analogs showed minimal VDR stimulating activity, their 1α-OH derivatives were potent VDR agonists. 4, 5, 14 and 24 significantly upregulated the expression of CYP24A1 at the mRNA level, consistent with their VDR activation abilities and indicating that 1α-hydroxylation is required to produce analogs with strong activity. These analogs have anti-inflammatory activities that are influenced by side chain composition and by 1α-hydroxylation. To understand their molecular interactions with the VDR, 20S(OH)D3, 4 and 33 were co-crystalized with the VDR ligand binding domain, which revealed subtle differences to the calcitriol-bound receptor. This study demonstrates the potential of the 20S(OH)D3 scaffold for the development of novel anti-inflammatory agents.

Investigation of the C-3-epi-25(OH)D3 of 25-hydroxyvitamin D3 in urban schoolchildren.

The physiological relevance C-3 epimer of 25-hydroxyvitamin D (3-epi-25(OH)D) is not well understood among youth. The objective of this study was to assess whether demographic/physiologic characteristics were associated with 3-epi-25(OH)D3 concentrations in youth. Associations between 3-epi-25(OH)D3 and demographics and between 3-epi-25(OH)D3, total 25-hydroxyvitamin (25(OH)D) (25(OH)D2 + 25(OH)D3), total cholesterol, high-density lipoprotein, low-density lipoprotein, and triglycerides were examined in racially/ethnically diverse schoolchildren (n = 682; age, 8-15 years) at Boston-area urban schools. Approximately 50% of participants had detectable 3-epi-25(OH)D3 (range 0.95-3.95 ng/mL). The percentage of 3-epi-25(OH)D3 of total 25(OH)D ranged from 2.5% to 17.0% (median 5.5%). Males were 38% more likely than females to have detectable 3-epi-25(OH)D3 concentrations. Both Asian and black race/ethnicity were associated with lower odds of having detectable 3-epi-25(OH)D3 compared with non-Hispanic white children (Asian vs. white, odds ratio (OR) 0.28, 95% confidence interval (CI) 0.14-0.53; black vs. white, OR 0.38, 95%CI 0.23-0.63, p < 0.001). Having an adequate (20-29 ng/mL) or optimal (>30 ng/mL) 25(OH)D concentration was associated with higher odds of having detectable 3-epi-25(OH)D3 than having an inadequate (<20 ng/mL) concentration (OR 4.78, 95%CI 3.23-6.94 or OR 14.10, 95%CI 7.10-28.0, respectively). There was no association between 3-epi-25(OH)D3 and blood lipids. However, when considering 3-epi-25(OH)D3 as a percentage of total 25(OH)D, total cholesterol was lower in children with percent 3-epi-25(OH)D3 above the median (mean difference -7.1 mg/dL, p = 0.01). In conclusion, among schoolchildren, sex, race/ethnicity, and total serum 25(OH)D concentration is differentially associated with 3-epi-25(OH)D. The physiological relevance of 3-epi-25(OH)D3 may be related to the 3-epi-25(OH)D3 as a percentage of total 25(OH)D and should be considered in future investigations.

Protein-ligand docking using FFT based sampling: D3R case study.

Fast Fourier transform (FFT) based approaches have been successful in application to modeling of relatively rigid protein-protein complexes. Recently, we have been able to adapt the FFT methodology to treatment of flexible protein-peptide interactions. Here, we report our latest attempt to expand the capabilities of the FFT approach to treatment of flexible protein-ligand interactions in application to the D3R PL-2016-1 challenge. Based on the D3R assessment, our FFT approach in conjunction with Monte Carlo minimization off-grid refinement was among the top performing methods in the challenge. The potential advantage of our method is its ability to globally sample the protein-ligand interaction landscape, which will be explored in further applications.

Nanofiber-based sutures induce endogenous antimicrobial peptide.

AIM: The aim of this study was to develop nanofiber-based sutures capable of inducing endogenous antimicrobial peptide production. METHODS: We used co-axial electrospinning deposition and rolling to fabricate sutures containing pam3CSK4 peptide and 25-hydroxyvitamin D3 (25D3). RESULTS: The diameters and mechanical properties of the sutures were adjustable to meet the criteria of United States Pharmacopeia designation. 25D3 exhibited a sustained release from nanofiber sutures over 4 weeks. Pam3CSK4 peptide also showed an initial burst followed by a sustained release over 4 weeks. The co-delivery of 25D3 and pam3CSK4 peptide enhanced cathelicidin antimicrobial peptide production from U937 cells and keratinocytes compared with 25D3 delivery alone. In addition, the 25D3/pam3CSK4 peptide co-loaded nanofiber sutures did not significantly influence proliferation of keratinocytes, fibroblasts, or the monocytic cell lines U937 and HL-60. CONCLUSION: The use of 25D3/pam3CSK4 peptide co-loaded nanofiber sutures could potentially induce endogenous antimicrobial peptide production and reduce surgical site infections.

1α,20S-Dihydroxyvitamin D3 Interacts with Vitamin D Receptor: Crystal Structure and Route of Chemical Synthesis.

1α,20S-Dihydroxyvitamin D3 [1,20S(OH)2D3], a natural and bioactive vitamin D3 metabolite, was chemically synthesized for the first time. X-ray crystallography analysis of intermediate 15 confirmed its 1α-OH configuration. 1,20S(OH)2D3 interacts with the vitamin D receptor (VDR), with similar potency to its native ligand, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] as illustrated by its ability to stimulate translocation of the VDR to the nucleus, stimulate VDRE-reporter activity, regulate VDR downstream genes (VDR, CYP24A1, TRPV6 and CYP27B1), and inhibit the production of inflammatory markers (IFNγ and IL1ß). However, their co-crystal structures revealed differential molecular interactions of the 20S-OH moiety and the 25-OH moiety to the VDR, which may explain some differences in their biological activities. Furthermore, this study provides a synthetic route for the synthesis of 1,20S(OH)2D3 using the intermediate 1α,3ß-diacetoxypregn-5-en-20-one (3), and provides a molecular and biological basis for the development of 1,20S(OH)2D3 and its analogs as potential therapeutic agents.

Structural insights into the mechanism of the drastic changes in enzymatic activity of the cytochrome P450 vitamin D3 hydroxylase (CYP107BR1) caused by a mutation distant from the active site.

Cytochromes P450 (P450s) are haem-containing enzymes that catalyze medically and industrially important oxidative reactions, and many P450s have been subjected to directed evolution and site-directed mutagenesis to improve their activity and substrate specificity. Nonetheless, in most cases the mechanism that leads to drastic changes in specific activity after the introduction of an amino-acid substitution distant from the active-site pocket is unclear. Here, two crystal structures of inactive mutants of the P450 vitamin D3 hydroxylase (Vdh), Vdh-F106V and Vdh-L348M, which were obtained in the course of protein-engineering experiments on Vdh, are reported. The overall structures of these mutants show an open conformation similar to that of wild-type Vdh (Vdh-WT), whereas a rearrangement of the common main-chain hydrogen bonds is observed in the CD-loop (residues 102-106), resulting in a more compactly folded CD-loop relative to that of Vdh-WT. The previously reported structures of Vdh-WT and of the highly active Vdh-T107A and Vdh-K1 mutants have a more stretched CD-loop, with partial formation of 310-helix-type hydrogen bonds, both in the open and closed states. Molecular-dynamics simulations also showed that the frequency of the 310-helix is significantly reduced in Vdh-F106V and Vdh-L348M. The closed conformation is crucial for substrate and ferredoxin binding to initiate the catalytic reaction of Vdh. Therefore, it is implied that the small local structural changes observed in this study might disrupt the conformational transition from the open to the closed state, thereby leading to a complete loss of vitamin D3 hydroxylase activity.

Investigating Differences in Gas-Phase Conformations of 25-Hydroxyvitamin D3 Sodiated Epimers using Ion Mobility-Mass Spectrometry and Theoretical Modeling.

Drift tube ion mobility coupled with mass spectrometry was used to investigate the gas-phase structure of 25-hydroxyvitamin D3 (25OHD3) and D2 (25OHD2) epimers, and to evaluate its potential in rapid separation of these compounds. Experimental results revealed two distinct drift species for the 25OHD3 sodiated monomer, whereas only one of these conformations was observed for its epimer (epi25OHD3). The unique species allowed 25OHD3 to be readily distinguished, and the same pattern was observed for 25OHD2 epimers. Theoretical modeling of 25OHD3 epimers identified energetically stable gas-phase structures, indicating that both compounds may adopt a compact "closed" conformation, but that 25OHD3 may also adopt a slightly less energetically favorable "open" conformation that is not accessible to its epimer. Calculated theoretical collision cross-sections for these structures agreed with experimental results to <2%. Experimentation indicated that additional energy in the ESI source (i.e., increased temperature, spray voltage) affected the ratio of 25OHD3 conformations, with the less energetically favorable "open" conformation increasing in relative intensity. Finally, LC-IM-MS results yielded linear quantitation of 25OHD3, in the presence of the epimer interference, at biologically relevant concentrations. This study demonstrates that ion mobility can be used in tandem with theoretical modeling to determine structural differences that contribute to drift separation. These separation capabilities provide potential for rapid (<60 ms) identification of 25OHD3 and 25OHD2 in mixtures with their epimers. Graphical Abstract ᅟ.

Development of an Improved Standard Reference Material for Vitamin D Metabolites in Human Serum.

The National Institute of Standards and Technology (NIST) has developed Standard Reference Material (SRM) 972a Vitamin D Metabolites in Frozen Human Serum as a replacement for SRM 972, which is no longer available. SRM 972a was developed in collaboration with the National Institutes of Health's Office of Dietary Supplements. In contrast to the previous reference material, three of the four levels of SRM 972a are composed of unmodified human serum. This SRM has certified and reference values for the following 25-hydroxyvitamin D [25(OH)D] species: 25(OH)D2, 25(OH)D3, and 3-epi-25(OH)D3. The value assignment and certification process included three isotope-dilution mass spectrometry approaches, with measurements performed at NIST and at the Centers for Disease Control and Prevention (CDC). The value assignment methods employed have been modified from those utilized for the previous SRM, and all three approaches now incorporate chromatographic resolution of the stereoisomers, 25(OH)D3 and 3-epi-25(OH)D3.