Loss of the glycosyltransferase Galnt11 affects vitamin D homeostasis and bone composition.

O-glycosylation is a conserved posttranslational modification that impacts many aspects of organismal viability and function. Recent studies examining the glycosyltransferase Galnt11 demonstrated that it glycosylates the endocytic receptor megalin in the kidneys, enabling proper binding and reabsorption of ligands, including vitamin D-binding protein (DBP). Galnt11-deficient mice were unable to properly reabsorb DBP from the urine. Vitamin D plays an essential role in mineral homeostasis and its deficiency is associated with bone diseases such as rickets, osteomalacia, and osteoporosis. We therefore set out to examine the effects of the loss of Galnt11 on vitamin D homeostasis and bone composition. We found significantly decreased levels of serum 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, consistent with decreased reabsorption of DBP. This was accompanied by a significant reduction in blood calcium levels and a physiologic increase in parathyroid hormone (PTH) in Galnt11-deficient mice. Bones in Galnt11-deficient mice were smaller and displayed a decrease in cortical bone accompanied by an increase in trabecular bone and an increase in a marker of bone formation, consistent with PTH-mediated effects on bone. These results support a unified model for the role of Galnt11 in bone and mineral homeostasis, wherein loss of Galnt11 leads to decreased reabsorption of DBP by megalin, resulting in a cascade of disrupted mineral and bone homeostasis including decreased circulating vitamin D and calcium levels, a physiological increase in PTH, an overall loss of cortical bone, and an increase in trabecular bone. Our study elucidates how defects in O-glycosylation can influence vitamin D and mineral homeostasis and the integrity of the skeletal system.

Plasma-based proteomic profiling identifies the distinct regulation of proteins in hyperplasia and endometrial cancer.

BACKGROUND: Among gynaecological malignancies, endometrial cancer (EC) is the most prevalent type of uterine cancer affecting women. This study explored the proteomic profiles of plasma samples obtained from EC patients, those with hyperplasia (Hy), and a control group (CO). A combination of techniques, such as 2D-DIGE, mass spectrometry, and bioinformatics, including pathway analysis, was used to identify proteins with modified expression levels, biomarkers and their associated metabolic pathways in these groups. METHODS: Thirty-four patients, categorized into three groups-10 with EC, 12 with Hy, and 12 CO-between the ages of 46 and 75 years old were included in the study. Untargeted proteomic analysis was carried out using two-dimensional difference in gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). RESULTS: In all three groups, 114 proteins that were significantly (p ≤ 0.05 and fold change ≥ 1.5) altered were successfully identified using peptide mass fingerprints (PMFs). Compared with those in the control group (CO), the EC samples had 85 differentially expressed proteins (39 upregulated and 46 downregulated), and in the Hy group, 81 proteins were dysregulated (40 upregulated and 41 downregulated) compared to those in the CO group, while 33 proteins exhibited differential regulation (12 upregulated and 21 downregulated) in the EC plasma samples compared to those in the Hy group. Vitamin D binding protein and complement C3 distinguished Hy and EC from CO with the greatest changes in expression. Among the differentially expressed proteins identified, enzymes with catalytic activity represented the largest group (42.9%). In terms of biological processes, most of the proteins were involved in cellular processes (28.8%), followed by metabolic processes (16.7%). STRING analysis for protein interactions revealed that the significantly differentially abundant proteins in the three groups are involved in three main biological processes: signalling of complement and coagulation cascades, regulation of insulin-like growth factor (IGF) transport and uptake by insulin-like growth factor binding proteins (IGFBPs), and plasma lipoprotein assembly, remodelling, and clearance. CONCLUSION: The identified plasma protein markers have the potential to serve as biomarkers for differentiating between EC and Hy, as well as for early diagnosis and monitoring of cancer progression.

The association between vitamin D binding protein levels and periodontal status: A systematic review.

BACKGROUND AND OBJECTIVES: Vitamin D binding protein (DBP) is biosynthesised in the liver and is predominantly expressed in serum. Its primary role centres on facilitating the systemic transportation of vitamin D and its metabolites, notably 25-hydroxyvitamin D, to specific target tissues where vitamin D exerts its biological functions. Due to the paucity of studies, it is unclear whether there is an association between DBP and periodontal status and thus its potential use as a diagnostic biomarker. Therefore, the aim of the systematic review is to investigate the association between DBP in periodontal disease. METHODS: Two independent reviewers (YD and RG) performed a systematic literature search of English publications using several databases including MEDLINE (OVID interface, 1946 onwards), EMBASE (OVID interface, 1974 onwards), and Global Health (OVID interface, 1973 onwards). This search strategy enabled the identification of relevant publications and the development of a comprehensive library of studies. Studies were included based on previously agreed eligibility criteria. Of the eight studies included as part of this systematic review, seven were case-control studies and one was a cross-sectional study. The quality assessment was based on the Newcastle-Ottawa Scale (NOS) for case-control studies and the modified NOS for the cross-sectional study. RESULTS: The NOS quality assessment was 'favorable' for 6 included case control studies; and 'fair' for one study. The modified NOS quality assessment for the single cross-sectional study demonstrated a medium risk of bias. The results of the majority of the included studies indicated a statistically significant higher concentration of DBP levels in individuals with periodontitis in comparison to those who were periodontally healthy. This trend held true irrespective of the sampling method employed for the assessment of DBP concentration. CONCLUSION: The results summarised in this systematic review indicate a positive association between DBP and periodontitis. Nonetheless, there is a need for longitudinal, prospective trials, to confirm the use of DBP as a potential biomarker for the diagnosis of periodontitis.

Evolution and impact of Standard Reference Materials (SRMs) for determining vitamin D metabolites.

The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health, Office of Dietary Supplements (NIH ODS), introduced the first Standard Reference Material® (SRM) for determining vitamin D metabolites in 2009 motivated by significant concerns about the comparability and accuracy of different assays to assess vitamin D status. After 14 years, a suite of five serum matrix SRMs and three calibration solution SRMs are available. Values were also assigned for vitamin D metabolites in five additional SRMs intended primarily to support measurements of other clinical diagnostic markers. Both the SRMs and the certification approach have evolved from significant exogenous serum content to primarily endogenous content and from value assignment by combining the results of multiple analytical methods to the use of measurements exclusively from reference measurement procedures (RMPs). The impact of the availability of these SRMs can be assessed by both the distribution information (sales) and by reports in the scientific literature describing their use for method validation, quality control, and research. In this review, we describe the development of these SRMs, the evolution in design and value assignment, the expansion of information reported, and SRM use in validating analytical methods and providing quality assurance within the vitamin D measurement community.

Parathyroidectomy reduces inflammatory cytokines and increases vitamin D metabolites in patients with primary hyperparathyroidism.

CONTEXT: Primary hyperparathyroidism (PHPT) is accompanied by a decreased 25-hydroxyvitamin D (25OHD) and vitamin D binding protein (DBP). High parathyroid hormone (PTH) is associated with elevated interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), yet the role of parathyroidectomy (PTX) on DBP and cytokines is not clear. OBJECTIVE: To prospectively evaluate the effect of PTX on inflammatory profiles, total and free 25OHD, and DBP in patients with PHPT. METHODS: Newly diagnosed patients with PHPT were recruited in the study (n=70). Twenty-eight patients returned after PTX, 3 months later. Biochemical markers were measured before and after PTX. A group of age and BMI-matched healthy subjects were included as controls (n=70). RESULTS: Prior to PTX, patients had lower serum DBP (37.5±6.0 vs 41.5±6.1mg/dL, p<0.001) and total 25OHD (30.1±9.5 vs 33.3±7.9ng/mL, P<0.05), but similar free 25OHD when compared to controls. Serum IL-6, C-reactive protein (CRP), and MCP-1 were higher in PHPT patients (p<0.05), whereas interleukin-10 (IL-10) was similar to controls. PTX increased total and free 25OHD and DBP (p<0.001), and decreased serum IL-6 and MCP-1 (p<0.05), but not CRP and IL-10. Multiple regression analysis indicated that the preoperative PTH explained a significant portion of the variance of IL-6 and MCP-1 (p<0.05). CONCLUSIONS: These findings suggest that PTH may upregulate the production of MCP-1 and IL-6 and downregulate circulating DBP in patients with PHPT, that are normalized by PTX. The exact effect of IL-6 and MCP-1 on DBP, vitamin D metabolites and the role on clinical outcomes in patients with PHPT is an area requiring further study.

Impact of Vitamin D deficiency on immunological and metabolic responses in women with recurrent pregnancy loss: focus on VDBP/HLA-G1/CTLA-4/ENTPD1/adenosine-fetal-maternal conflict crosstalk.

BACKGROUND AND AIM: Recurrent pregnancy loss (RPL), also known as recurrent implantation failure (RIF), is a distressing condition affecting women characterized by two or more consecutive miscarriages or the inability to carry a pregnancy beyond 20 weeks. Immunological factors and genetic variations, particularly in Vit D Binding Protein (VDBP), have gained attention as potential contributors to RPL. This study aimed to provide insight into the immunological, genetic, and metabolic networks underlying RPL, placing a particular emphasis on the interactions between VDBP, HLA-G1, CTLA-4, ENTPD1, and adenosine-fetal-maternal conflict crosstalk. METHODS: A retrospective study included 198 women with three or more consecutive spontaneous abortions. Exclusion criteria comprised uterine abnormalities, endocrine disorders, parental chromosomal abnormalities, infectious factors, autoimmune diseases, or connective tissue diseases. Immunological interplay was investigated in 162 female participants, divided into two groups based on their Vit D levels: normal Vit D-RPL and low Vit D-RPL. Various laboratory techniques were employed, including LC/MS/MS for Vit D measurement, ELISA for protein detection, flow cytometry for immune function analysis, and molecular docking for protein-ligand interaction assessment. RESULTS: General characteristics between groups were significant regarding Vit D and glucose levels. Low Vit D levels were associated with decreased NK cell activity and downregulation of HLA-G1 and HLA-G5 proteins, while CTLA-4 revealed upregulation. VDBP was significantly downregulated in the low Vit D group. Our findings highlight the intricate relationship between Vit D status and adenosine metabolism by the downregulation of SGLT1, and NT5E, key components of adenosine metabolism, suggests that Vit D deficiency may disrupt the regulation of adenosine levels, leading to an impaired reproductive outcome. HNF1ß, a negative regulator of VDBP, was upregulated, while HNF1α, a positive regulator, was downregulated in low Vit D women after RPL. Molecular docking analysis revealed crucial residues involved in the interaction between Vit D and HNF1ß. CONCLUSION: Collectively, these findings underscore the importance of Vit D in modulating immune function and molecular pathways relevant to pregnancy maintenance, highlighting the need for further research to elucidate the mechanisms and potential therapeutic interventions for improving pregnancy outcomes in individuals with Vit D deficiency and RPL.

Phenomewide Association Study of Health Outcomes Associated With the Genetic Correlates of 25 Hydroxyvitamin D Concentration and Vitamin D Binding Protein Concentration.

While it is known that vitamin D deficiency is associated with adverse bone outcomes, it remains unclear whether low vitamin D status may increase the risk of a wider range of health outcomes. We had the opportunity to explore the association between common genetic variants associated with both 25 hydroxyvitamin D (25OHD) and the vitamin D binding protein (DBP, encoded by the GC gene) with a comprehensive range of health disorders and laboratory tests in a large academic medical center. We used summary statistics for 25OHD and DBP to generate polygenic scores (PGS) for 66,482 participants with primarily European ancestry and 13,285 participants with primarily African ancestry from the Vanderbilt University Medical Center Biobank (BioVU). We examined the predictive properties of PGS25OHD, and two scores related to DBP concentration with respect to 1322 health-related phenotypes and 315 laboratory-measured phenotypes from electronic health records. In those with European ancestry: (a) the PGS25OHD and PGSDBP scores, and individual SNPs rs4588 and rs7041 were associated with both 25OHD concentration and 1,25 dihydroxyvitamin D concentrations; (b) higher PGS25OHD was associated with decreased concentrations of triglycerides and cholesterol, and reduced risks of vitamin D deficiency, disorders of lipid metabolism, and diabetes. In general, the findings for the African ancestry group were consistent with findings from the European ancestry analyses. Our study confirms the utility of PGS and two key variants within the GC gene (rs4588 and rs7041) to predict the risk of vitamin D deficiency in clinical settings and highlights the shared biology between vitamin D-related genetic pathways a range of health outcomes.

Vitamin D and reproductive disorders: a comprehensive review with a focus on endometriosis.

Vitamin D is a fat-soluble steroid hormone that was initially known only for regulating calcium and phosphorus levels and maintaining bone health. However, it was later discovered that many organs express vitamin D metabolizing enzymes and have a ligand for vitamin D, which regulates the expression of an extensive assortment of genes. As a result, vitamin D is indispensable for the proper function of organs, and its deficiency is believed to be a critical factor in symptoms and disorders such as cardiovascular diseases, autoimmune diseases, and cancers. The significance of vitamin D in reproductive tissues was recognized later, and studies have revealed its crucial role in male and female fertility, as well as proper reproductive function during pregnancy. Vitamin D deficiency has been identified as a risk factor for infertility, gonadal cancers, pregnancy complications, polycystic ovary syndrome, and endometriosis. However, data investigating the association between vitamin D levels and reproductive disorders, including endometriosis, have encountered inconsistencies. Therefore, the present study aims to review existing research on the effect of vitamin D on proper reproductive function, and the role of deficiency in reproductive diseases and specifically focuses on endometriosis.

Investigating Vitamin D-Binding Protein's Role in Childhood Health and Development.

Vitamin D-binding protein (DBP), also known as Gc-globulin, is a protein that affects several physiological processes, including the transport and regulation of vitamin D metabolites. Genetic polymorphisms in the DBP gene have a significant impact on vitamin D levels and may have implications for disease risk. DBP polymorphisms are linked to differential immune responses, which could influence the onset of juvenile diseases. This narrative review examines the various roles of DBP, with a focus on bone health, immunological regulation, and lipid metabolism in children. Chronic disorders affected by DBP polymorphisms include bone abnormalities, autoimmune diseases, cardiovascular issues, childhood asthma, allergies, cystic fibrosis, acute liver failure, celiac disease, inflammatory bowel disease, and chronic kidney disease. Future research should focus on identifying the processes that underpin the many roles that DBP plays and developing customized therapeutics to improve health outcomes in the juvenile population.

The Effect of Phototherapy on Systemic Inflammation Measured with Serum Vitamin D-Binding Protein and hsCRP in Patients with Inflammatory Skin Disease.

Vitamin D plays a role in inflammatory skin disease, but the exact mechanisms and the clinical significance remain unclear. According to the free hormone hypothesis, it is the free concentration of 25-hydroxy vitamin D (25(OH)D) that is biologically active. Vitamin D-binding protein (DBP) acts as the major transporter of vitamin D in the circulation, and DBP concentration defines the free 25(OH)D levels. DBP levels are elevated in various inflammatory conditions, including psoriasis. Narrowband-ultraviolet B (NB-UVB) is the most widely used phototherapy and is an established first-line treatment for psoriasis and atopic dermatitis (AD), often used before proceeding to systemic treatment. The aim of this study was to investigate the influence of NB-UVB phototherapy on DBP and high-sensitivity C-reactive protein (hsCRP) levels, as markers of systemic inflammation, in inflammatory skin disease. Thirty adults (psoriasis (n = 20) and AD (n = 10)) were treated with NB-UVB. Serum DBP, hsCRP, total and free 25(OH)D, and 1,25-dihydroxy vitamin D (1,25(OH)2D) were measured before and after NB-UVB. Disease severity was assessed with Psoriasis Area and Severity Index (PASI), SCORing Atopic Dermatitis (SCORAD), and Visual Analogue Scale (VAS). DBP decreased in psoriasis patients and varied with no clear trend in AD patients. HsCRP decreased in both groups, but this did not reach statistical significance. PASI, SCORAD, and VAS improved, and vitamin D levels increased after NB-UVB. Sub-analysis indicated a better response to NB-UVB for patients with vitamin D deficiency and insufficiency compared to vitamin D-sufficient patients. The decrease in DBP after NB-UVB in psoriasis patients suggests a potential systemic anti-inflammatory effect of phototherapy. Measurement of vitamin D levels may potentially serve as a tool to identify patients who would derive the greatest benefit from NB-UVB phototherapy.

Vitamin D/Bone Mineral Density and Triglyceride Paradoxes Seen in African Americans: A Cross-Sectional Study and Review of the Literature.

Vitamin D is known to have a positive effect on bone health. Despite the greater frequency of vitamin D deficiency in African Americans (AA), they have a higher bone mineral density (BMD) compared to whites, demonstrating a disconnect between BMD and vitamin D levels in AA. Another intriguing relationship seen in AA is the triglyceride (TG) paradox, an unusual phenomenon in which a normal TG status is observed even when patients house conditions known to be characterized by high TG levels, such as Type II diabetes. To the best of our knowledge, no study has examined whether these two paradoxical relationships exist simultaneously in AA subjects with Type II diabetes. In this study, we compared levels of blood markers, including HbA1c, TG, and vitamin D, measured as serum 25-hydroxyvitamin D [25(OH)VD] µM/mL, [25(OH)VD]/TG, calcium, and BMD in AA (n = 56) and white (n = 26) subjects with Type II diabetes to see whether these relationships exist concurrently. We found that AA subjects had significantly lower TG and [25(OH)VD] levels and a significantly higher BMD status compared to white subjects, even when the ages, BMI, duration of diabetes, HbA1c, and calcium levels were similar between the two groups. This demonstrates that these two paradoxical relationships exist simultaneously in Type II diabetic AA subjects. In addition to these findings, we discuss the current hypotheses in the literature that attempt to explain why these two intriguing relationships exist. This review also discusses four novel hypotheses, such as altered circulating levels and the potential role of estrogen and hydrogen sulfide on BMD and HMG-CoA reductase as a possible contributor to the TG paradox in AA subjects. This manuscript demonstrates that there are still many unanswered questions regarding these two paradoxical relationships and further research is needed to determine why they exist and how they can be implemented to improve healthcare.

Genetic Variants in Vitamin-D Metabolism Genes (rs1155563, rs12785878 and rs10500804) among Females with Type-2 Diabetes Mellitus in Saudi Arabia.

Background & Objective: Hypovitaminosis D has shown to be linked with T2DM development and control in numerous studies. The association of SNPs in genes related to VitD metabolism with T2DM has not been sufficiently studied. Consequently, our aim in the present study was to explore the association between genetic variants in genes connected with VitD, mainly a SNP in GC (rs1155563), a SNP in DHCR7 (rs12785878) and a SNP in CYP2R1 (rs10500804) with glycaemic parameters in females with T2DM in Saudi Arabia. Methods: The cross-sectional study included 149 females (age 38-52 years) with T2DM from Jeddah, Saudi Arabia (September 2022-March 2023). Blood was extracted from the participants for biochemical tests including measuring VitD [25(OH)D] concentration, parameters of glycaemia (HbA1c, insulin, fasting glucose and insulin sensitivity indices including HOMA2-IR and HOMA2-%ß), and for genomic DNA isolation. Sanger DNA sequencing was used to screen for VitD genetic polymorphisms (rs1155563, rs12785878 and rs10500804). Results: Minor allele frequency for rs1155563C, rs12785878T and rs10500804G was 0.21, 0.23 and 0.37, respectively. Levels of 25(OH)D and glycaemic parameters as well did not show any significant difference between the genotypes of each SNP. Conclusion: This study showed lack of association of rs1155563 in GC, rs12785878 in DHCR7 and rs10500804 in CYP2R1 with VitD level primarily and with glycaemic parameters secondarily. Additional research is required to explore further other VitD genetic polymorphisms influencing T2DM which might lead consequently to genetically-based personalized management for T2DM.

Links Between Vitamin D-Binding Protein, Alpha-1 Antitrypsin and Neutrophil Proteins in Meconium.

BACKGROUND/AIMS: Alpha-1 antitrypsin (AAT), vitamin-D binding protein (VDBP) and neutrophil granule proteins are specifically related to the neutrophil function and may be considered candidate biomarkers detected and measured in meconium (the first feces of newborn infants) as signals indicating abnormal processes in the fetal stage. Individual proteins found in meconium can be a source of information pertaining to the intrauterine metabolic processes. METHODS: Concentrations of AAT, VDBP, calprotectin, myeloperoxidase, lactoferrin and elastase were measured using ELISA tests in 80 meconium samples collected from 19 healthy, full-term neonates. RESULTS: The meconium concentrations of VDBP and AAT (mean±SD, [mg/g meconium]: 3.74±6.93, 3.72±1.79, respectively) were approximately 1000 times higher than those of the protein granule proteins calprotectin, myeloperoxidase, elastase and lactoferrin (mean ± SD, [µg/g meconium]: 285.7±215.8, 1.83±1.73, 1.72±2.70, 45.58±78.89, respectively). The correlation between VDBP and AAT was negative (r= - 0.40. p=0.000) and those between VDBP and calprotectin (r=0.38, p=0.000) and VDBP and myeloperoxidase (r=0.45, p=0.000) were positive. AAT was found to correlate positively with lactoferrin (r=0.38, p=0.000). CONCLUSION: The correlations between the concentrations of VDBP and AAT, and with neutrophil granule proteins observed in meconium indicate their functional relationship in the intrauterine environment of the developing fetus. Meconium can be seen as an apparently underutilized source of biomarkers for evaluation of metabolic processes specific to fetal development.

Increased Serum Total and Free 25-Hydroxyvitamin D with Daily Intake of Cholecalciferol-Fortified Skim Milk: A Randomized Controlled Trial in Colombian Adolescents.

BACKGROUND: The efficacy of cholecalciferol (vitamin D3) food fortification in low- and middle-income countries near the Equator is unknown. OBJECTIVES: We examined the effects of providing cholecalciferol-fortified skim milk to adolescents and their mothers on serum total 25(OH)D, free 25(OH)D, and vitamin D-binding protein (DBP) concentrations in a randomized controlled trial. METHODS: We randomly assigned 80 Colombian families each with a child aged 12-14.5 y and their mother 1 L of skim milk daily, either fortified with 2400 IU (60 µg) cholecalciferol or unfortified, for 6 wk. We prescribed 500 mL of milk daily to adolescents; mothers consumed the remainder ad libitum. We estimated intent-to-treat effects as the between-arm difference in the change in serum total and free 25(OH)D and DBP concentrations from baseline to the end of follow-up. Secondary analyses included stratification by baseline characteristics and per-protocol comparisons. RESULTS: Among adolescents, fortification effects (95% CI) on serum total 25(OH)D, free 25(OH)D, and DBP concentrations were 5.4 nmol/L (2.1, 8.8 nmol/L), 0.6 pmol/L (-0.2, 1.4 pmol/L), and -416 nmol/L (-944, 112 nmol/L), respectively. Effects on total 25(OH)D were stronger in adolescents with lower DBP concentrations, darker skin, less sunlight exposure, and higher compliance than in their respective counterparts. Fortification increased free 25(OH)D concentrations in high compliers. Among mothers, the effects (95% CI) on total 25(OH)D and DBP concentrations were 4.0 nmol/L (0.6, 7.5 nmol/L) and -128 nmol/L (-637, 381 nmol/L), respectively. There were no adverse events. CONCLUSIONS: Provision of cholecalciferol-fortified skim milk increases serum total 25(OH)D concentrations in Colombian adolescents and adult women.

A review of the critical role of vitamin D axis on the immune system.

In recent years, the physiological and molecular functions of vitamin D (Vit-D) have been deeply investigated. At first, Vit-D was considered a regulator of mineral and skeletal homeostasis. However, due to the extensive-expression pattern of Vit-D receptor (VDR) in almost every non-skeletal cell, Vit-D is considered mainly a multifunctional agent with broad effects on various tissues, notably the immune system. The expression of VDR in immune cells such as dendritic cells, monocyte/macrophage, neutrophils, B cells and T cells has been well demonstrated. Besides, such immune cells are capable of metabolizing the active form of Vit-D which means that it can module the immune system in both paracrine and autocrine manners. Vit-D binding protein (DBP), that regulates the levels and homeostasis of Vit-D, is another key molecule capable of modulating the immune system. Recent studies indicate that dysregulation of Vit-D axis, variations in the DBP and VDR genes, and Vit-D levels might be risk factors for the development of autoimmune disease. Here, the current evidence regarding the role of Vit-D axis on the immune system, as well as its role in the development of autoimmune disease will be clarified. Further insight will be given to those studies that investigated the association between single nucleotide polymorphisms of DBP and VDR genes with autoimmune disease susceptibility.

The influence of proteoforms: assessing the accuracy of total vitamin D-binding protein quantification by proteolysis and LC-MS/MS.

OBJECTIVES: Vitamin D-binding protein (VDBP), a serum transport protein for 25-hydroxyvitamin D [25(OH)D], has three common proteoforms which have co-localized amino acid variations and glycosylation. A monoclonal immunoassay was found to differentially detect VDBP proteoforms and methods using liquid chromatography-tandem mass spectrometry (LC-MS/MS) might be able to overcome this limitation. Previously developed multiple reaction monitoring LC-MS/MS methods for total VDBP quantification represent an opportunity to probe the potential effects of proteoforms on proteolysis, instrument response and quantification accuracy. METHODS: VDBP was purified from homozygous human donors and quantified using proteolysis or acid hydrolysis and LC-MS/MS. An interlaboratory comparison was performed using pooled human plasma [Standard Reference Material® 1950 (SRM 1950) Metabolites in Frozen Human Plasma] and analyses with different LC-MS/MS methods in two laboratories. RESULTS: Several shared peptides from purified proteoforms were found to give reproducible concentrations [≤2.7% coefficient of variation (CV)] and linear instrument responses (R2≥0.9971) when added to human serum. Total VDBP concentrations from proteolysis or amino acid analysis (AAA) of purified proteoforms had ≤1.92% CV. SRM 1950, containing multiple proteoforms, quantified in two laboratories resulted in total VDBP concentrations with 7.05% CV. CONCLUSIONS: VDBP proteoforms were not found to cause bias during quantification by LC-MS/MS, thus demonstrating that a family of proteins can be accurately quantified using shared peptides. A reference value was assigned for total VDBP in SRM 1950, which may be used to standardize methods and improve the accuracy of VDBP quantification in research and clinical samples.

Association between genetic variants in key vitamin-D-pathway genes and external apical root resorption linked to orthodontic treatment.

This study evaluated the association between single-nucleotide polymorphisms (SNPs) in vitamin-D-related genes and the amount of external apical root resorption linked to orthodontic treatment. One hundred and forty-three individuals were assessed. The amount of external apical root resorption of upper central incisors (EARRinc ) and lower first molars (EARRmol ) were evaluated in radiographs. Seven SNPs were genotyped across four genes including the vitamin D receptor [VDR], group-specific component [GC], cytochrome P450 family 27 subfamily B member 1 [CYP27B1], and cytochrome P450 family 24 subfamily A member 1 [CYP24A1]. Linear regressions were implemented to determine allele-effects on external apical root resorption. Individuals carrying the AA genotype in VDR rs2228570 had a 21% higher EARRmol than those having AG and GG genotypes (95% CI: 1.03,1.40). EARRmol in heterozygous rs2228570, was 12% lower than for homozygotes (95%CI: 0.78,0.99). Participants with the CCG haplotype (rs1544410-rs7975232-rs731236) in VDR had an EARRmol 16% lower than those who did not carry this haplotype. Regarding CYP27B1 rs4646536, EARRinc in participants who had at least one G allele was 42% lower than for homozygotes AA (95%CI: 0.37,0.93). Although these results did not remain significant after multiple testing adjustment, potential associations may still be suggested. Further replication studies are needed to confirm or refute these findings.

Vitamin D and bone metabolism in Graves' disease: a prospective study.

PURPOSE: Vitamin D and osteoporosis in Graves' disease (GD) have been examined in cross-sectional studies with divergent results. Here, we prospectively studied vitamin D metabolism and bone health in patients with newly diagnosed GD. METHODS: Thirty consecutive patients with de novo overt thyrotoxicosis diagnosed with GD were included. At diagnosis, none of the patients were treated with vitamin D or anti-osteoporotic drugs. All patients were initially treated with antithyroid drugs. Blood samplings were taken at baseline and at 6 weeks, 3, 6, 12 and 24 months after treatment start. Serum levels of 25OHD3, 1,25OH2D3, calcium, parathyroid hormone (PTH), and C-terminal telopeptides of Type I collagen (CTX-I) were analysed. Bone mineral density (BMD) was measured at baseline, and 1 and 2 years after treatment initiation. RESULTS: At diagnosis, patients with GD did not have vitamin D deficiency. There were no significant correlations between levels of 25OHD3 and thyrotoxicosis. Upon treatment of the thyrotoxicosis, serum calcium fell transiently, and PTH and 1,25OH2D3 increased. 25OHD3 fell within the normal range and stabilised at 6 months. CTX-I fell over 12 months, BMD increased significantly up to 2 years, p = 0.002, < 0.001 and 0.005 in the spine, left total hip and left femoral neck, respectively. CONCLUSIONS: The present data underline that thyrotoxicosis has a negative impact on bone health and demonstrate fine-tuned dynamics in bone and vitamin D metabolism. Upon treatment, bone health improved over a follow-up period of 24 months despite rising PTH. Increased conversion of 25OHD3 to 1,25OH2D3 occurs during treatment of GD.

Vitamin D receptor and vitamin D binding protein gene polymorphisms in patients with asthma: a pilot study.

BACKGROUND: The effects of vitamin D are exerted by interaction with the vitamin D receptor (VDR) and vitamin D binding protein (VDBP). Polymorphisms in VDR or VDBP genes may affect vitamin D levels, influencing the pathogenesis of asthma and atopy. The aim of this study was to investigate the possible association of VDR and VDBP gene single-nucleotide polymorphisms (SNP), 25-hydroxyvitamin D (25(OH)D), blood eosinophils and total IgE level in subjects with asthma in comparison with healthy individuals. METHODS: This case-control study enrolled 63 subjects with asthma (45 allergic and 18 non-allergic) and 32 healthy subjects were involved in the study. Sensitization of subjects to inhaled allergens was determined by a skin prick test, lung function was evaluated by spirometry. Blood eosinophil count was determined by standard methods. Serum 25(OH)D and total IgE levels were evaluated by ELISA. Polymorphisms in the VDR and VDBP genes on the 12q13.11 and 4q13.3 chromosomal region were analyzed using TaqMan SNP Genotyping Assay probes. RESULTS: In asthma patients with vitamin D deficiency (< 20 ng/ml) the allele G of rs11168293 of VDR was more common than in those having insufficiency (20-30 ng/ml) of vitamin D (63% and 31%, p < 0.05). Moreover, asthmatic subject with rs11168293 G allele has significant higher blood eosinophil count compared to asthmatic without the rs11168293 G allele (8.5 ± 12.3% vs. 5.1 ± 1.5%, p < 0.05). Significantly higher IgE level was found in subjects with allergic asthma with the allele A of rs7041 on VDBP gene than in those without this allele (540 ± 110 and 240 ± 80 IU/ml, p < 0.05). CONCLUSIONS: The association of polymorphisms in VDBP and VDR gene, the rs11168293 G allele and the rs7041 A allele, with 25(OH)D, blood eosinophil and total IgE level in asthma, let us suggest that vitamin D, VDR and VDBP gene polymorphisms are important in pathogenesis of asthma despite its form in relation to atopy.

The role of free vitamin D and vitamin D binding protein in SARS-Cov-2 infection in children.

BACKGROUND: Many studies have discussed the effects of serum vitamin D deficiency in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) patients. This study aimed to investigate the relationship between SARS-CoV-2 infection severity and free vitamin D (FVD) and bioavailable vitamin D (BAVD) levels in children. METHODS: A prospective case-control study design was used. Participants were divided into three groups based on the World Health Organization COVID-19 Clinical Progression Scale. Serum 25-hydroxyvitamin D (ng/mL), albumin (g/L), and vitamin D binding protein (ng/mL) levels were evaluated to investigate the relationship between disease severity and FVD and BAVD levels. RESULTS: In total, 82 participants were included in the study. Of those, 24.4% were uninfected (n = 20), 50% had a mild case of SARS-CoV-2 (n = 41), and 25.6% had a moderate case (n = 21). There was a statistically significant difference in FVD and BAVD levels between the groups (p = 0.026). Median FVD (p = 0.007, Cohen's d = 0.84) and BAVD (p = 0.007, Cohen's d = 0.86) levels were significantly higher in the mild group compared to the moderate group. FVD and BAVD metabolites were moderately positively correlated with lymphocyte counts (FVD: r = 0.437, p < 0.001; BAVD: r = 0.439, p < 0.001). CONCLUSIONS: This is the first study to demonstrate a relationship between SARS-CoV-2 symptom severity and FVD and BAVD levels. The relationship between FVD and BAVD levels and lymphocyte counts could play an important role in symptom severity and should be evaluated in further studies.