Free 25-hydroxyvitamin Research Articles

The Expression of Vitamin D Receptor on Peripheral Blood Mononuclear Cells in Patients with Psoriasis.

Vitamin D plays an important role in psoriasis, but its involvement in pathogenesis remains unclear. This study aimed to evaluate vitamin D receptor (VDR) expression on peripheral blood mononuclear cells (PBMCs) in patients with psoriasis and healthy controls and to study the effects of the Etanercept treatment on VDR expression on PBMCs in patients with psoriasis. Twenty patients with moderate to severe psoriasis received treatment with Etanercept for 24 weeks. The age- and sex-matched controls did not receive any intervention. VDR expression on CD3+ lymphocytes and CD14+ monocytes, and serum levels of total and free 25-hydroxyvitamin D (25(OH)D) and 1,25-dihydroxy vitamin D (1,25(OH)2D) were analyzed at baseline, after 10-12 weeks, and after 24 weeks in both groups. VDR expression was analyzed using flow cytometry. We observed higher expression of the VDR on CD14+ monocytes in psoriasis patients compared to healthy controls at baseline. This difference was no longer significant after 24 weeks of the Etanercept treatment. The patients with psoriasis improved clinically. However, VDR expression was unaltered during the Etanercept treatment, and there was no correlation between VDR expression and disease severity.

Investigation of the Relationship between Vitamin D Deficiency and Vitamin D-Binding Protein Polymorphisms in Severe COVID-19 Patients.

This study explores the association of vitamin D-binding protein (VDBP) gene polymorphisms, vitamin D levels, and the severity of COVID-19, including the need for intensive care unit (ICU) hospitalization. We analyzed a cohort of 56 consecutive age- and gender-matched adult COVID-19-positive patients and categorized them into three groups: outpatients with mild illness, inpatients with moderate disease, and ICU patients. We measured levels of free, total, and bioavailable 25-hydroxyvitamin D [25(OH)D], VDBP, and albumin. VDBP polymorphisms rs5488 and rs7041 were identified using real-time PCR. A significant proportion of ICU patients were vitamin D-deficient (56.25%) compared to outpatients (10%) and inpatients (5%) (p = 0.0003). ICU patients also had notably lower levels of VDBP (median: 222 mg/L) and total 25(OH)D (median: 18.8 ng/mL). Most patients carried heterozygous rs7041 (60.7%) and wild-type rs4588 (58.9%) genotypes. The distribution of rs7041 SNP varied significantly among groups (p = 0.0301), while rs4588 SNP distribution did not (p = 0.424). Heterozygous rs4588 patients had significantly lower VDBP levels (p = 0.029) and reduced bioavailable 25(OH)D compared to those with wild-type rs4588 (p = 0.020). Our findings indicate that VDBP gene polymorphisms, particularly rs7041 and rs4588, are associated with vitamin D status and the severity of COVID-19. The lower VDBP levels and bioavailable vitamin D in ICU patients suggest that these genetic variants may influence disease severity and hospitalization needs. These results highlight the potential role of VDBP polymorphisms in COVID-19 severity, suggesting that genetic screening could be valuable in assessing the risk of severe outcomes and guiding personalized treatment strategies.

Effects of Supplemental Vitamin D3, Omega-3 Fatty Acids on Physical Performance Measures in VITamin D and OmegA-3 TriaL.

Declining muscle strength and performance in older adults are associated with falls, fractures, and premature death. To determine whether supplementation with vitamin D3 or omega-3 fatty acids vs. placebo for 2 years improves physical performance measures. VITamin D and OmegA-3 TriaL (VITAL) was a double-blinded, placebo-controlled randomized trial of supplemental vitamin D3 and/or omega-3 fatty acids vs. placebo in the prevention of cancer and cardiovascular disease in 25,871 U.S. adults. This ancillary study was completed in a New England sub-cohort that had in-person evaluations at baseline and 2-year follow-up. Center for Clinical Investigations in Boston. 1,054 participants (men ≥50 and women ≥55 years). 2x2 factorial design of supplemental vitamin D3 (cholecalciferol, 2000 IU/day) and/or marine omega-3 fatty acids (1 g/day). 2-year changes in physical performance measures of grip strength, walking speed, standing balance, repeated chair stands, and Timed-up and Go (TUG). At 2 years, all randomized groups showed worsening walking speeds and TUG. There were no differences in changes in grip strength, walking speeds, Short Physical Performance Battery (composite of walking speed, balance, and chair stands), and TUG between the vitamin D3-treated and the placebo-treated groups and between the omega-3-treated and the placebo-treated groups. Effects overall did not vary by sex, age, body mass index, or baseline measures of total or free 25-hydroxyvitamin D (25[OH]D) or plasma n-3 index; TUG slightly worsened with vitamin D supplementation, compared to placebo, in participants with baseline total 25(OH)D levels above the median (p=0.01, p for interaction=0.04). Neither supplemental vitamin D3 nor marine omega-3 fatty acids for 2 years improved physical performance in this generally healthy adult population.

Associations between total, free and bioavailable 25-hydroxyvitamin D forms with adiponectin and irisin in maternal-neonatal pairs at birth from Greece.

Apart from the well-established skeletal effects, vitamin D has been explored as a secretagogue influencing various adipokines, including adiponectin and irisin. Recent evidence suggests that specific forms of 25-Hydroxyvitamin D (25(OHD), such as free and bioavailable 25(OH)D, may provide more accurate measurements of vitamin D status. The relationship between vitamin D status and serum irisin and adiponectin concentrations remains largely unexplored, particularly during pregnancy. We analyzed data from 67 healthy maternal-neonatal pairs from Northern Greece at birth. Biochemical and hormonal tests were conducted on each maternal-neonatal pair. The vitamin D forms were estimated using validated mathematical models. Subsequently, regression analyses were conducted to determine the association between the vitamin D forms and adipokine levels. Bioavailable maternal 25(OH)D was inversely associated with neonatal irisin concentrations [β=-73.46 (-140.573 to -6.341), p=0.034]. No other associations were observed between maternal vitamin D status and neonatal adipokine concentrations. In conclusion, maternal bioavailable vitamin D concentrations are inversely associated with neonatal serum irisin concentrations, warranting further studies to evaluate the underlying mechanisms for this finding.

Vitamin D and asthma in children: insights from total and free 25-hydroxyvitamin D and vitamin D binding protein measurements

Vitamin D and asthma in children: insights from total and free 25-hydroxyvitamin D and vitamin D binding protein measurements

Associations of free, bioavailable and total 25-hydroxyvitamin D with neonatal birth anthropometry and calcium homoeostasis in mother-child pairs in a sunny Mediterranean region.

Sufficient vitamin D status is crucial for successful pregnancy and fetal development. The assessment of 25-hydroxyvitamin D (25(OH)D) concentrations is commonly used to evaluate vitamin D status. Our objective was to examine the interrelated biodynamics of maternal and neonatal total, free and bioavailable 25(OH)D in maternal-neonatal dyads at birth and their associations with homeostasis and neonatal birth anthropometry. We analysed a cohort of seventy full-term mother-child pairs. We found positive associations between all neonatal measures of vitamin D status. Maternal forms exhibited a similar pattern of association, except for the bioavailable maternal form. In multivariate analysis, both total and free maternal 25(OH)D concentrations were correlated with all neonatal forms (neonatal total 25(OH)D: 1·29 (95 % CI, 1·12, 1·46) for maternal total 25(OH)D, 10·89 (8·16, 13·63) for maternal free 25(OH)D), (neonatal free 25(OH)D: 0·15 for maternal total 25(OH)D, 1·28 (95 % CI, 0·89, 1·68) for maternal free 25(OH)D) and (0·13 (95 % CI, 0·10, 0·16), 1·06 (95 % CI, 0·68, 1·43) for maternal free 25(OH)D), respectively, with the exclusion of the bioavailable maternal form. We observed no significant interactions within or between groups regarding maternal and neonatal vitamin D parameters and maternal calciumand parathyroid hormone concentrations, and neonatal birth anthropometry. Our study indicates that bioavailable maternal and neonatal 25(OH)D have no significant effects on vitamin D equilibrium, Ca homeostasis and neonatal anthropometry at birth. However, we observed an interaction between maternal and neonatal total and free 25(OH)D concentrations at the maternal-neonatal interface, with no associations observed with other calciotropic or anthropometric outcomes.

Free and bioavailable 25-hydroxyvitamin D thresholds for bone metabolism and their associations with metabolic syndrome in Chinese women of childbearing age.

The free hormone hypothesis suggests that free and bioavailable 25-hydroxyvitamin D [25(OH)D] may better reflect vitamin D bioactivity. This study aimed to determine the free and bioavailable 25(OH)D characteristics, estimate their thresholds based on parathyroid hormone (PTH) and bone turnover markers (BTMs), assess their associations with the risk of metabolic syndrome (MetS), and evaluate their potential advantages. A cross-sectional study was conducted using a nationally representative database (n = 1,505, female, 18-45 years). Serum total 25(OH)D, vitamin D-binding protein, albumin, PTH, and BTMs [osteocalcin, β-CrossLaps of type 1 collagen containing cross-linked C-telopeptide (β-CTX), and procollagen type 1 N-terminal propeptide (P1NP)] were measured. Free 25(OH)D and bioavailable 25(OH)D were calculated. The threshold associations of 25(OH)D with PTH and BTMs were analyzed. The relationship between 25(OH)D and MetS risk was examined. An intervention study was then performed in 39 women (18-47 years) to assess the associations of increasing 25(OH)D with PTH and BTMs after vitamin D supplementation. In the cross-sectional study, the three forms of 25(OH)D were found to have similar distribution characteristics. Free and bioavailable 25(OH)D correlated well with total 25(OH)D. Significant total 25(OH)D cutoffs were observed for PTH (14.19 ng/mL and 18.03 ng/mL), osteocalcin (15.14 ng/mL), β-CTX (14.79 ng/mL), and P1NP (15.08 ng/mL). Free and bioavailable 25(OH)D cutoffs were only found for P1NP (3.47 pg/mL and 1.66 ng/mL, respectively). A total 25(OH)D of <15.14 ng/mL was marginally associated with a higher risk of reduced high-density lipoprotein cholesterol (HDL-C) [odd ratios (OR) = 1.371 (0.991-1.899)]. The ORs of higher versus lower free and bioavailable 25(OH)D levels for reduced HDL-C were 0.770 (0.621-0.956) and 0.772 (0.622-0.958), respectively. The results of the intervention study indicated that PTH and BTMs responded more sensitively to total 25(OH)D than to free or bioavailable 25(OH)D. Free and bioavailable 25(OH)D only had a threshold effect on P1NP. The active 25(OH)D thresholds could be used for risk assessment of reduced HDL-C. However, no superiority of free or bioavailable 25(OH)D was found based on the response of PTH and BTMs to changes in 25(OH)D in Chinese women of childbearing age following vitamin D supplementation. http://www.chictr.org.cn, ChiCTR2200058290.

Total and free 25-hydroxyvitamin D concentrations and risk of urinary incontinence in women participating in Nurses Health Studies I & II

BackgroundUrgency urinary incontinence (UUI) occurs in >40% of older women. Our objective was to examine the relationship of total and free plasma 25-hydroxyvitamin D (25(OH)D) and UUI to evaluate vitamin D status as a novel target for prevention of UUI. MethodsWe conducted a nested case control study using the Nurses’ Health Study (NHS) and NHS II. Using stored plasma samples from 2000, we measured total 25(OH)D, free 25(OH)D, and intact parathyroid hormone (PTH) levels and examined their relationship to incident UUI from 2000 to 2013. Plasma biomarker levels were categorized as quartiles. Multivariable-adjusted odds ratios of UUI were estimated by conditional logistic regression models (with matching by age) across categories of each biomarker and covariates. ResultsThe analytic sample included 398 cases of incident UUI and 398 matched controls with a mean age of 50 years. We found a strong correlation of plasma levels of total 25(OH)D with free 25(OH)D (r=0.5). Plasma total 25(OH)D and free 25(OH)D concentrations were negatively correlated with PTH (r=-0.08 AND -0.09, respectively). Overall, we found no evidence that levels of total plasma 25(OH)D, free 25(OH)D, or PTH were related to incident UUI after adjustment for obesity, physical activity, cigarette smoking, menopausal status, hypertension, and type 2 diabetes. ConclusionsFree plasma 25(OH)D by quartile, as well as total plasma 25(OH)D, was not associated with incident UUI in women. We found that plasma total and free 25(OH)D were highly correlated with each other and inversely correlated with PTH. Plasma free 25(OH)D did not provide additional predictive value in determining risk of UUI.

Vitamin D Status Determines the Impact of Metformin on Gonadotropin Levels in Postmenopausal Women.

Metformin was found to decrease elevated levels of anterior pituitary hormones. Its impact on lactotrope secretory function was absent in women with vitamin D insufficiency. This study investigated whether vitamin D status determines metformin action on overactive gonadotropes. We compared the effect of six-month metformin treatment on the plasma levels of gonadotropins, TSH, prolactin, ACTH, estradiol, free thyroid hormones, IGF-1, and 25-hydroxyvitamin D, as well as on glucose homeostasis markers between three matched groups of postmenopausal women at high risk for diabetes: untreated subjects with vitamin D insufficiency (group A), untreated women with normal vitamin D status (group B), and individuals receiving vitamin D supplementation with normal 25-hydroxyvitamin D levels (group C). Only in groups B and C did metformin reduce FSH levels and tend to decrease LH levels, and these effects correlated with baseline gonadotropin levels, baseline 25-hydroxyvitamin D levels, and the improvement in insulin sensitivity. Follow-up gonadotropin levels were higher in group A than in the other two groups. The drug did not affect circulating levels of TSH, prolactin, ACTH, estradiol, free thyroid hormones, IGF-1, or 25-hydroxyvitamin D. The obtained results suggest that the impact of metformin on gonadotropin secretion in women after menopause is determined by vitamin D status.

Effect of supplementation of vitamin D3 or vitamin D2 on serum concentrations of free and total 25-hydroxyvitamin D and the expression of genes involved in immune function in peripheral blood mononuclear cells of weaned pigs

ABSTRACT The present study aimed to compare the effects of vitamin D2 and vitamin D3 supplementation on concentrations of total and free 25(OH)D in plasma and the expression of genes involved in the innate immune system in peripheral blood mononuclear cells (PBMC) in weaned pigs. Five groups of pigs (with an initial body weight of around 9 kg) received basal diets supplemented with either 500 (control group), 1000 or 2000 IU vitamin D3/kg diet or 1000 or 2000 IU vitamin D2/kg diet for a period of 4 weeks. Vitamin D supplementation did not influence feed intake, body weight gain, feed conversion ratio, apparent total tract digestibility of calcium and phosphorus, and serum concentrations of calcium, inorganic phosphate and parathyroid hormone. Supplementation of vitamin D3 led to a dose-dependent increase of the concentrations of total and free 25(OH)D in serum. In contrast, pigs supplemented with 1000 or 2000 IU vitamin D2/kg diet did not have higher concentrations of total and free 25(OH)D in serum than the control group. The ratio of free/total 25(OH)D in serum was not influenced by vitamin D3 supplementation, whereas the group supplemented with 2000 IU vitamin D2/kg diet had a higher free/total 25(OH)D ratio than the groups supplemented with 1000 or 2000 IU vitamin D3/kg diet. Genes involved in vitamin D signalling (CYP27B1, VDR), as well as pro-inflammatory and immune regulatory genes (TLR4, TNF, IL1B and TGFB1) and genes encoding porcine protegrins (NPG1, NPG4), proteins belonging to the group of antimicrobial peptides, in PBMC were not different among groups supplemented with vitamin D3 or vitamin D2 and the control group. Therefore, the study indicates that supplementation of vitamin D2 causes much lower levels of total 25(OH)D than supplementation of vitamin D3 and that supplementation of vitamins D2 or D3 at moderate levels does not have an impact on the innate immune function in healthy pigs.

Variations in plasma free 25-hydroxyvitamin D concentrations during intermittent fasting are independently correlated with amino acid intake among meat and non-meat eaters

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Correction: Vičič et al. Assessment of Vitamin D Status in Slovenian Premenopausal and Postmenopausal Women, Using Total, Free, and Bioavailable 25-Hydroxyvitamin D (25(OH)D). Nutrients 2022, 14, 5349

In the original publication [...]

Different patterns of changes in free 25-hydroxyvitamin D concentrations during intermittent fasting among meat eaters and non-meat eaters and correlations with amino acid intake

We prospectively assessed changes in free 25-hydroxyvitamin D [25(OH)D] and vitamin D binding protein (VDBP) among overweight adults who followed a pescatarian Orthodox intermittent fasting regimen (n = 59) and controls who followed a low-fat 12:12 diet (n = 46). Total and free 25(OH)D, parathyroid hormone, VDBP, anthropometric data, and amino acid intake were evaluated in both groups at three time points: at baseline, 7 weeks after diet implementation, and 5 weeks after participants returned to their usual eating habits (12 weeks from baseline). An increase in amino acid intake between baseline and 12 weeks was independently correlated with higher free 25(OH)D values at 12 weeks for both groups. Our findings suggest that diet can affect free 25(OH)D concentrations, through variations in amino acid intake, independently of exposure to sunlight, providing novel mechanistic insights into the future planning of vitamin D supplementation strategies. However, this hypothesis needs to be tested in larger studies.

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

BackgroundThe efficacy of cholecalciferol (vitamin D3) food fortification in low- and middle-income countries near the Equator is unknown. ObjectivesWe 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. MethodsWe 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. ResultsAmong 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. ConclusionsProvision of cholecalciferol-fortified skim milk increases serum total 25(OH)D concentrations in Colombian adolescents and adult women.

Total, free, and bioavailable 25-hydroxyvitamin D levels in patients with periodontitis (stage III): a case-control study.

The aim of this study was to investigate the relationship between total, free and bioavailable 25-hydroxyvitamin D levels, and periodontitis. Thirty-nine patients with periodontitis diagnosis and 44 healthy participants were included in this study. 25-Hydroxyvitamin D, vitamin D-binding protein, procalcitonin, and albumin levels were measured from the serum samples obtained. Free and bioavailable 25-hydroxyvitamin D levels were calculated. Total, free, and bioavailable 25-hydroxyvitamin D levels were compared between the healthy control and periodontitis groups. The association of total 25 (OH)D levels with periodontitis was tested through univariate analysis and multivariate logistic regression. Total 25-hydroxyvitamin D, bioavailable 25-hydroxyvitamin D, and free 25-hydroxyvitamin D levels were significantly lower in the periodontitis group than in the healthy control group (p < 0.001). In the logistic regression analysis, a significant correlation was observed between total 25-hydroxyvitamin D and periodontitis (p<0.001). Our study shows that there is a relationship between total, free and bioavailable 25-hydroxyvitamin D levels and periodontitis. In addition, it has been shown that the determination of free and bioavailable 25-hydroxyvitamin D levels does not provide additional advantagesin terms of the relationship between periodontitis and vitamin D. In this case-control study, we examined the relationship between total, free and bioavailable 25-hydroxyvitamin D and periodontitis. We found lower total, free, and bioavailable 25-hydroxyvitamin D levels in periodontitis patients. It was also found that vitamin D-binding protein may be an important biomarker in the identification of various forms of periodontal disease. ClinicalTrials.gov Identifier NCT05343273.

Assessment of Vitamin D Status in Slovenian Premenopausal and Postmenopausal Women, Using Total, Free, and Bioavailable 25-Hydroxyvitamin D (25(OH)D).

The objective of our study was to evaluate vitamin D status and its predictors in Slovenian premenopausal and postmenopausal women. A cross-sectional study was carried out between 1 March 2021 and 31 May 2021. A total of 319 healthy women from the Central Slovenian region aged between 44 and 65 were recruited; 176 were included in the final analysis. The vitamin D status was determined by measuring the total 25-Hydroxycholecalciferol (25(OH)D) concentration, vitamin D binding protein (DBP), and albumin and calculating the bioavailable 25(OH)D and free 25(OH)D. For the calculation of bioavailable and free 25(OH)D, we developed a new online calculator. The Endocrine Society's thresholds for vitamin D deficiency and insufficiency were used; 29.0% of premenopausal and 24.4% of postmenopausal subjects were found to be vitamin D deficient (total 25(OH)D &lt; 50 nmol/L); 76.8% of the premenopausal and 61.7% of postmenopausal subjects were found to have insufficient levels (total 25(OH)D &lt; 75 nmol/L). Premenopausal women had 11.8% lower total 25(OH)D, 32.2% lower bioavailable 25(OH)D, and 25.2% higher DBP than postmenopausal women. The most important predictors of vitamin D status were vitamin D supplementation and time spent in the sun. Contrary to similar studies, the vitamin D status in Slovenian postmenopausal women was significantly better than in premenopausal women. In postmenopausal women, the measurement of free or bioavailable 25(OH)D instead of the total 25(OH)D could be advantageous.

Serum TSH, 25(OH) D and phosphorus levels predict weight loss in individuals with diabetes/prediabetes and morbid obesity: a single-center retrospective cohort analysis

BackgroundTo evaluate the association of vitamin D and thyroid-stimulating hormone (TSH) with weight loss (WL) percentage (%) in patients with diabetes/prediabetes and Class II/III obesity.MethodsA retrospective cohort study was designed. Data were collected from a database of a referral endocrinology clinic that is prospectively and systematically generated. After exclusion of unavailable cases, the study enrolled 285 patients (51 ± 11 years old, female/male = 208/77; diabetes/prediabetes = 159/126; no/on levothyroxine replacement = 176/109; Class II/III obesity = 184/101, respectively) who maintained euthyroidism and were followed up for ≥6 months. The data were analyzed to determine the predictors of WL%.ResultsCompared with baseline, in the median 22 months of follow-up, the whole study group lost 5.1% of their baseline body weight. As most obesity management trials define success as ‘at least 10% of WL compared to baseline’, we stratified the patients based on WL% extents. The distribution was as follow: Group 1 (n = 61) lost ≥10% body weight, Group 2 (n = 162) lost < 10% body weight, while Group 3 (n = 62) gained weight by the final visit. In groups 1 and 2 (weight losers), the serum thyroid stimulatig hormone (TSH) and parathyroid hormone (PTH) levels decreased and the free thyroxine (fT4), calcium, phosphorus, and 25-hydroxyvitamin D (25(OH)D) levels increased. In Group 3 (weight gainers), these changes were not observed (except for an increase in calcium levels). Regression analysis revealed that the final visit TSH (β = − 0.14, p < 0.05), 25(OH) D (β = 0.15, p < 0.05), and phosphorus (β = 0.20, p < 0.05) levels predicted WL%. However, if patients with autoimmune thyroiditis were excluded from the analysis, the decrease in TSH levels was not statistically significant.ConclusionsSerum TSH, phosphorus, and 25(OH) D levels predict WL% in euthyroid patients with diabetes/prediabetes and morbid obesity. TSH predictivity seems to be a function of thyroid autoimmunity present with increased frequency in this cohort. Greater levels of phosphorus within the reference range and a sufficient vitamin D status are associated with a greater WL%.

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.

Associations between serum vitamin D biomarkers and tumor expression of Ki67, p53, and COX-2 in colorectal cancer cases from the Southern Community Cohort Study

Higher 25-hydroxyvitamin D is associated with lower colorectal cancer (CRC) risk, with limited data from African Americans (AAs), who have greater risk for CRC and 25-hydroxyvitamin D deficiency. In a predominantly AA sample of CRC cases from the Southern Community Cohort Study (SCCS), we report associations between vitamin D biomarkers and tumor expression of proteins implicated in vitamin D’s anti-tumorigenic pathways (e.g. proliferation and inflammation) and CRC prognosis. SCCS participants with incident CRC were identified via state cancer registries. Serum 25-hydroxyvitamin D and vitamin D binding protein (VDBP) were measured at enrollment. ‘Free’ 25-hydroxyvitamin D was calculated via standard equation. Cellular Ki67, p53, and COX-2 were measured from tumor samples and categorized using literature-defined cut-points related to survival. Generalized linear models were used to measure associations between vitamin D exposures, tumor biomarkers, and stage. In total, 104 cases (40–79 years) were analyzed. 25-hydroxyvitamin D was not associated with high Ki67 (odds ratio (OR) per 1-standard deviation (SD) increase [95% confidence interval] 1.35[0.86–2.11]), p53 (0.75[0.47–1.20]), or COX-2 expression (1.25[0.78–2.01]), or metastatic disease (1.04[0.59–1.81]). Mean biomarker expression was unrelated to 25-hydroxyvitamin D (p-trend ≥.09). Null associations were observed for VDBP and free 25-hydroxyvitamin D. In AAs (n = 70), higher VDBP was associated with lower odds of high Ki67 expression (0.53[0.28–0.98], p-trend =.04). In conclusion, we observed no associations between 25-hydroxyvitamin D and prognostic marker expression in CRC. An inverse association between VDBP and tumor Ki67 in AAs is consistent with reports showing relationships with reduced CRC mortality.

Gluten-free diet attenuates the impact of exogenous vitamin D on thyroid autoimmunity in young women with autoimmune thyroiditis: a pilot study

Although both exogenous vitamin D and a gluten-free diet were found to reduce thyroid antibody titers, no study investigated interactions between gluten intake and vitamin D status in patients with autoimmune thyroid disorders. The aim of the present study was to assess whether the gluten-free diet determines the effect of vitamin D treatment on thyroid autoimmunity and thyroid function in young women with autoimmune (Hashimoto’s) thyroiditis. The study compared two groups of euthyroid premenopausal women with this disorder, matched for thyroid antibody titers: 31 women with non-celiac gluten sensitivity complying for at least 12 months with the gluten-free diet and 31 unaffected sisters of women with non-celiac gluten sensitivity remaining without any dietary intervention. Plasma titers of thyroid peroxidase and thyroglobulin antibodies, as well as plasma concentrations of thyrotropin, free thyroid hormones, prolactin, 25-hydroxyvitamin D and high-sensitive C-reactive protein were measured at entry and after a six-month follow-up. Moreover, at both time points, the structure parameters of thyroid homeostasis were assessed. Although exogenous vitamin D decreased titers of thyroid peroxidase and thyroglobulin antibodies and increased 25-hydroxyvitamin D levels in each treatment group, this effect was less pronounced in patients on the gluten-free diet than in patients not following any dietary recommendations. Only in the latter group of patients, vitamin D increased SPINA-GT. Treatment-induced changes in thyroid peroxidase and thyroglobulin antibodies correlated with the impact of treatment on 25-hydroxyvitamin D levels. The obtained results suggest that gluten-free diet may impair beneficial effects of exogenous vitamin D in individuals with Hashimoto’s thyroiditis.