Evaluation of vitamin D3 metabolites in Callithrix jacchus (common marmoset).

Vitamin D3 (cholecalciferol) is endogenously produced in the skin of primates when exposed to the appropriate wavelengths of ultraviolet light (UV-B). Common marmosets (Callithrix jacchus) maintained indoors require dietary provision of vitamin D3 due to lack of sunlight exposure. The minimum dietary vitamin D3 requirement and the maximum amount of vitamin D3 that can be metabolized by marmosets is unknown. Observations of metabolic bone disease and gastrointestinal malabsorption have led to wide variation in dietary vitamin D3 provision amongst research institutions, with resulting variation in circulating 25-hydroxyvitamin D3 (25(OH)D3 ), the accepted marker for vitamin D sufficiency/deficiency. Multiple studies have reported serum 25(OH)D3 in captive marmosets, but 25(OH)D3 is not the final product of vitamin D3 metabolism. In addition to serum 25(OH)D3, we measured the most physiologically active metabolite, 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3 ), and the less well understood metabolite, 24,25-dihydroxyvitamin D3 (24,25(OH)2 D3 ) to characterize the marmoset's ability to metabolize dietary vitamin D3 . We present vitamin D3 metabolite and related serum chemistry value colony reference ranges in marmosets provided diets with 26,367 (Colony A, N = 113) or 8,888 (Colony B, N = 52) international units (IU) of dietary vitamin D3 per kilogram of dry matter. Colony A marmosets had higher serum 25(OH)D3 (426 ng/ml [SD 200] vs. 215 ng/ml [SD 113]) and 24,25(OH)2 D3 (53 ng/ml [SD 35] vs. 7 ng/ml [SD 5]). There was no difference in serum 1,25(OH)2 D3 between the colonies. Serum 1,25(OH)2 D3 increased and 25(OH)D3 decreased with age, but the effect was weak. Marmosets tightly regulate metabolism of dietary vitamin D3 into the active metabolite 1,25(OH)2 D3 ; excess 25(OH)D3 is metabolized into 24,25(OH)2 D3 . This ability explains the tolerance of high levels of dietary vitamin D3 by marmosets, however, our data suggest that these high dietary levels are not required.

Increase of 1,25 dihydroxyvitamin D in sarcoidosis patients with renal dysfunction.

INTRODUCTION: In sarcoidosis, renal involvement includes hypercalcemia-related nephrocalcinosis and granulomatous tubulointerstitial nephritis. Hypercalcemia is thought to be due to increased production of 1,25 dihydroxyvitamin D (1-25D), but 1-25D levels have not been evaluated in sarcoidosis patients with renal dysfunction. MATERIALS AND METHODS: We enrolled 9 sarcoidosis patients who underwent renal biopsy, and compared the serum 1-25D concentration and eGFR with those in 428 non-sarcoidosis patients who had renal dysfunction (stage 2 or higher CKD with an estimated glomerular filtration rate < 90). RESULTS: Serum calcium and 1-25D levels were significantly higher in the sarcoidosis patients than in the non-sarcoidosis patients (p < 0.01 and p = 0.01, respectively). There was a positive correlation between 1-25D and eGFR in the patients without sarcoidosis (r = 0.693; p < 0.01). As the renal function of sarcoidosis patients was improved by steroid therapy, the serum 1-25D and adjusted serum calcium levels decreased to near the median values in non-sarcoidosis patients. On renal biopsy, CD68 staining was positive for tissue macrophages in all 8 patients who had tubulointerstitial nephritis (with or without typical granulomas), while Von Kossa staining showed calcification of tubules near or inside granulomas in 6 of these 8 patients. CONCLUSION: While tissue macrophages promote development of tubulointerstitial nephritis and 1-25D overproduction in renal sarcoidosis, hypercalcemia secondary to elevation of 1-25D may be related to renal calcification and granuloma formation.

Ancestry-Adjusted Vitamin D Metabolite Concentrations in Association With Cytochrome P450 3A Polymorphisms.

We investigated the association between genetic polymorphisms in cytochrome P450 (CYP2R1, CYP24A1, and the CYP3A family) with nonsummer plasma concentrations of vitamin D metabolites (25-hydroxyvitamin D3 (25(OH)D3) and proportion 24,25-dihydroxyvitamin D3 (24,25(OH)2D3)) among healthy individuals of sub-Saharan African and European ancestry, matched on age (within 5 years; n = 188 in each ancestral group), in central suburban Pennsylvania (2006-2009). Vitamin D metabolites were measured using high-performance liquid chromatography with tandem mass spectrometry. Paired multiple regression and adjusted least-squares mean analyses were used to test for associations between genotype and log-transformed metabolite concentrations, adjusted for age, sex, proportion of West-African genetic ancestry, body mass index, oral contraceptive (OC) use, tanning bed use, vitamin D intake, days from summer solstice, time of day of blood draw, and isoforms of the vitamin D receptor (VDR) and vitamin D binding protein. Polymorphisms in CYP2R1, CYP3A43, vitamin D binding protein, and genetic ancestry proportion remained associated with plasma 25(OH)D3 after adjustment. Only CYP3A43 and VDR polymorphisms were associated with proportion 24,25(OH)2D3. Magnitudes of association with 25(OH)D3 were similar for CYP3A43, tanning bed use, and OC use. Significant least-squares mean interactions (CYP2R1/OC use (P = 0.030) and CYP3A43/VDR (P = 0.013)) were identified. A CYP3A43 genotype, previously implicated in cancer, is strongly associated with biomarkers of vitamin D metabolism. Interactive associations should be further investigated.

The vitamin D metabolite ratio (VMR) as a predictor of functional biomarkers of bone health.

CONTEXT: The vitamin D metabolite ratio (VMR) (serum 24,25(OH)2 D3 /25(OH)D3 ) has been proposed as a biomarker of vitamin D sufficiency to replace serum 25(OH)D. OBJECTIVE: To examine the relationships of 24,25(OH)2 D3 and VMR to functional biomarkers of bone health following vitamin D supplementation. SETTING: An ambulatory research centre. DESIGN: Serum from a previous research study of dose response of PTH, calcium absorption and bone turnover to vitamin D supplementation was analysed for vitamin D metabolites (25(OH)D, 24,25(OH)2 D3 ). OUTCOME: The relationship of serum 24,25(OH)2 D3 and VMR to calcium absorption, PTH and bone turnover markers was examined. RESULTS: Although there were strong correlations of serum 25(OH)D with 24,25(OH)2 D3 and free 25(OH)D, its correlation with VMR was lower. After vitamin D supplementation, the change in 25(OH)D, 24,25(OH)2 D3 and VMR was associated with the change in calcium absorption, PTH and CTX. The correlation of the change in PTH with the change in metabolites was the lowest for VMR. Moreover, estimated dose response for standardized values of vitamin D metabolites showed a beta-coefficient for VMR that was significantly less in magnitude compared to other metabolites. CONCLUSION: Serum 24,25(OH)2 D3 is closely associated with the dose response of serum 25(OH)D to vitamin D supplementation. However, the VMR does not appear to be equivalent to either of these metabolites in its response to increasing vitamin D intake or its association with PTH. It is unlikely that VMR will replace 25(OH)D as a biomarker for vitamin D sufficiency.

Use of Extended-Release Calcifediol to Treat Secondary Hyperparathyroidism in Stages 3 and 4 Chronic Kidney Disease.

BACKGROUND/AIMS: Vitamin D insufficiency and secondary hyperparathyroidism (SHPT) are associated with increased morbidity and mortality in chronic kidney disease (CKD) and are poorly addressed by current treatments. The present clinical studies evaluated extended-release (ER) calcifediol, a novel vitamin D prohormone repletion therapy designed to gradually correct low serum total 25-hydroxyvitamin D, improve SHPT control and minimize the induction of CYP24A1 and FGF23. METHODS: Two identical multicenter, randomized, double-blind, placebo-controlled studies enrolled subjects from 89 US sites. A total of 429 subjects, balanced between studies, with stage 3 or 4 CKD, SHPT and vitamin D insufficiency were randomized 2:1 to receive oral ER calcifediol (30 or 60 µg) or placebo once daily at bedtime for 26 weeks. Most subjects (354 or 83%) completed dosing, and 298 (69%) entered a subsequent open-label extension study wherein ER calcifediol was administered without interruption for another 26 weeks. RESULTS: ER calcifediol normalized serum total 25-hydroxyvitamin D concentrations (>30 ng/ml) in >95% of per-protocol subjects and reduced plasma intact parathyroid hormone (iPTH) by at least 10% in 72%. The proportion of subjects receiving ER calcifediol who achieved iPTH reductions of ≥30% increased progressively with treatment duration, reaching 22, 40 and 50% at 12, 26 and 52 weeks, respectively. iPTH lowering with ER calcifediol was independent of CKD stage and significantly greater than with placebo. ER calcifediol had inconsequential impact on serum calcium, phosphorus, FGF23 and adverse events. CONCLUSION: Oral ER calcifediol is safe and effective in treating SHPT and vitamin D insufficiency in CKD.

Development and validation of the simultaneous measurement of four vitamin D metabolites in serum by LC-MS/MS for clinical laboratory applications.

The quantification of serum 25-hydroxyvitamin D [25(OH)D] as an indicator of vitamin D status is currently primarily conducted by immunoassays, yet LC-MS/MS would allow more accurate determination. Furthermore, LC-MS/MS would allow simultaneous measurement of multiple analytes. The aim of this study was to develop and validate an LC-MS/MS method to simultaneously measure four vitamin D metabolites (25(OH)D3, 3-epi-25(OH)D3, 25(OH)D2, and 24,25(OH)2D3) in serum for clinical laboratory applications. Serum samples were first prepared in a 96-well supported liquid extraction plate and the eluate was derivatized using the Cookson-type reagent 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD), which rapidly and quantitatively reacts with the s-cis-diene structure of vitamin D metabolites. The derivatized samples were subjected to LC-MS/MS, ionized by electrospray ionization (positive-ion mode), and detected by selected reaction monitoring. The lower limits of quantification for 25(OH)D3, 3-epi-25(OH)D3, 25(OH)D2, and 24,25(OH)2D3 were 0.091, 0.020, 0.013, and 0.024 ng/mL, respectively. The accuracy values and the extraction recoveries for these four metabolites were satisfactory. Serum 25(OH)D levels determined by our LC-MS/MS were compared with those obtained by conventional radioimmunoassay (RIA) that cannot distinguish 25(OH)D3 and 25(OH)D2. The values obtained by the RIA method exhibited a mean bias of about 8.35 ng/mL, most likely as a result of cross reaction of the antibody with low-abundance metabolites, including 24,25(OH)2D3. Various preanalytical factors, such as long sample sitting prior to serum separation, repeated freeze-thaw cycles, and the presence of anticoagulants, had no significant effects on these determinations. This high-throughput LC-MS/MS simultaneous assay of the four vitamin D metabolites 25(OH)D3, 3-epi-25(OH)D3, 25(OH)D2, and 24,25(OH)2D3 required as little as 20 µL serum. This method will aid further understanding of low-abundance vitamin D metabolites, as well as the accurate determination of 25(OH)D3 and 25(OH)D2.

Dynamics of Vitamin D Metabolism in Maternal-Fetal Dyads.

OBJECTIVES: Metabolites of vitamin D in maternal-neonatal dyads remain relatively unexplored. The goal of this study was to evaluate concentrations of 25(OH)D3, 24,25(OH)2D3, and 3-epi-25(OH)D3 in maternal-infant pairs at delivery. METHODS: Serum samples of maternal and infant cord blood were collected on 131 mother-infant pairs at delivery. Vitamin D metabolites were analyzed in triplicate using liquid chromatography-tandem mass spectrometry. Statistical analysis was conducted using the Fisher exact test, Wilcoxon rank sum test, and Spearman correlation coefficients. RESULTS: Mean 25(OH)D3 concentrations in maternal and cord blood were 32.9 and 18.5 ng/mL, respectively; mean maternal and cord 24,25(OH)2D3 were 2.0 versus 1.1 ng/mL, respectively. Absolute concentrations of 3-epi-25(OH)D3 were similar in maternal and cord samples (2.4 vs 2.2 ng/mL), whereas the proportion of the total 25(OH)D as the 3-epimer was 6.5% in maternal samples and 10.5% in cord samples. This suggests that the fetus contributes significantly to 3-epi-25(OH)D3 production. In contrast, the ratio of 25(OH)D3:24,25(OH)2D3 was identical in maternal and cord samples (18.5) suggesting equivalent CYP24A1 activity in mother and fetus. Maternal and cord metabolite levels were highly correlated (r = 0.78, 0.90, 0.89 for 25(OH)D3, 24,25(OH)2D3, and 3-epi-25(OH)D3, respectively, P = 0.001 for all). Serum concentrations of all metabolites were lower in nonwhite infants compared with white infants. Maternal and cord concentrations of 25(OH)D3 were positively associated with birth weight (r = 0.21, P = 0.02; r = 0.25, P = 0.003, respectively). CONCLUSIONS: This data suggests that although maternal and cord concentrations of vitamin D metabolites are highly correlated, regulation of specific vitamin D metabolites in the mother and the neonate may be mediated independently.

Effect of Antiepileptic Therapy on Serum 25(OH)D3 and 24,25(OH)2D3 Levels in Epileptic Children.

BACKGROUND: Vitamin D deficiency is not only associated with the adverse effects of chronic treatment with antiepileptic drugs (AEDs), but also with epilepsy. Although emerging evidence suggests that AEDs can accelerate the vitamin D catabolism, resulting in suboptimal vitamin D status, there are a limited number of studies examining the vitamin D status in epileptic patients, especially in first-episode or AEDs-naïve children. METHODS: Determined with high-performance liquid chromatography-tandem mass spectrometry, circulating 25(OH)D3 and 24,25(OH)2D3 levels, and 24,25(OH)2D3:25(OH)D3 ratio were compared between AEDs-treated epileptic (n = 363) and control (n = 159) children. To further figure out whether the patients were in a vitamin D deficient prone state even before treatment, epileptic children before their initiation of treatment (n = 51) were enrolled into a follow-up study. RESULTS: A significant decrease of 25(OH)D3 and 24,25(OH)2D3 levels, but a significant increase of 24,25(OH)2D3:25(OH)D3 ratio was observed in epileptic children, compared with controls. Baseline 25(OH)D3, 24,25(OH)2D3 and 24,25(OH)2D3:25(OH)D3 ratio in the follow-up group were similar to those in controls, but significantly changed with 2 months of AED therapy. CONCLUSIONS: Disturbed vitamin D levels were possibly the consequence of AED therapy, rather than the contributing factor of epilepsy. Collectively, circulating vitamin D levels should be monitored and corrected in AEDs-treated epileptic children.

Candidate Reference Measurement Procedure for the Determination of (24R),25-Dihydroxyvitamin D3 in Human Serum Using Isotope-Dilution Liquid Chromatography-Tandem Mass Spectrometry.

The two major forms of vitamin D, vitamin D3 and vitamin D2, are metabolized in the liver through hydroxylation to 25-hydroxyvitamin D species, and then further hydroxylated in the kidney to various dihydroxyvitamin D species. (24R),25-Dihydroxyvitamin D3 ((24R),25(OH)2D3) is a major catabolite of 25-hydroxyvitamin D metabolism and is an important vitamin D metabolite used as a catabolism marker and indicator of kidney disease. The National Institute of Standards and Technology has recently developed a reference measurement procedure for the determination of (24R),25(OH)2D3 in human serum using isotope-dilution LC-MS/MS. The (24R),25(OH)2D3 and added deuterated labeled internal standard (24R),25(OH)2D3-d6 were extracted from serum matrix using liquid-liquid extraction prior to LC-MS/MS analysis. Chromatographic separation was performed using a fused-core C18 column. Atmospheric pressure chemical ionization in the positive ion mode and multiple reaction monitoring were used for LC-MS/MS. The accuracy of the measurement of (24R),25(OH)2D3 was evaluated by recovery studies of measuring (24R),25(OH)2D3 in gravimetrically prepared spiked samples of human serum with known (24R),25(OH)2D3 levels. The recoveries of the added (24R),25(OH)2D3 averaged 99.0% (0.8% SD), and the extraction efficiencies averaged 95% (2% SD). Excellent repeatability was demonstrated with CVs of ∼1%. The limit of quantitation at a signal-to-noise ratio of ∼10 was 0.2 ng/g. Potential isomeric interferences from other endogenous species and from impurity components of the reference standard were investigated. LC baseline resolution of (24R),25(OH)2D3 from these isomers was achieved within 35 min. This method was used for value assignment of (24R),25(OH)2D3 in Standard Reference Materials of Vitamin D Metabolites in Human Serum, which can serve as an accuracy base for routine methods used in clinical laboratories.

Significance of serum 24,25-dihydroxyvitamin D in the assessment of vitamin D status: a double-edged sword?

BACKGROUND: 24,25-Dihydroxyvitamin D [24,25(OH)2D] in serum may be both a nuisance and nutritionally valuable. METHODS: We investigated the impact of 24,25(OH)2D3 on the performance of commercially available immunoassays for serum total 25-hydroxyvitamin D [25(OH)D] using (a) serum from a nationally representative sample of adults, (b) serum from a spiking experiment, and (c) data from the UK Vitamin D External Quality Assurance Scheme (DEQAS). We also investigated the utility of the serum ratio of 24,25(OH)2D3 to 25(OH)D as an index of inactivation and of response to vitamin D supplementation using randomized controlled trial (RCT) data. Measurement of 24,25(OH)2D in sera by a LC-MS/MS method allowed for an investigation of its impact on immunoassay-derived serum 25(OH)D values as well as its clinical utility. We report data from a nationally representative sample of adults, a recent vitamin D RCT in older adults, and DEQAS. RESULTS: 24,25(OH)2D3 contributed to the positive bias observed in some immunoassays relative to LC-MS/MS-derived estimates for total 25(OH)D. A spiking experiment showed that the degree of cross-reactivity with 24,25(OH)2D was high and may underpin this positive bias. Adjustment for 24,25(OH)2D3 concentration brought estimates closer to true values. Data from the vitamin D RCT showed that the ratio of 24,25(OH)2D3 to 25(OH)D was associated with serum 25(OH)D3 and with response of serum 25(OH)D to vitamin D supplementation. CONCLUSIONS: Our findings highlight that the effect of 24,25(OH)2D3 in serum is a double-edged sword-an interferent for some immunoassays, yet potentially informative of nutritional status.

24,25-Dihydroxyvitamin d3 and vitamin D status of community-dwelling black and white Americans.

BACKGROUND: 24,25-Dihydroxyvitamin D [24,25(OH)2D] is a metabolite of 25-hydroxyvitamin D (25D). Blacks frequently have low total 25D without manifestations of vitamin D deficiency, suggesting that total serum 25D may incorrectly reflect vitamin D status in different racial groups. The ratio of serum 24,25(OH)2D to 25D [vitamin D metabolite ratio (VMR)] represents a new candidate biomarker for vitamin D status. METHODS: We measured 24,25(OH)2D3 and 25D3 by mass spectrometry in a random community cohort of black (n = 212) and white (n = 164) Americans to evaluate VMR as a marker for vitamin D status. We measured parathyroid hormone concentrations by immunoassay to compare VMR and 25D3 against a physiological indicator of vitamin D deficiency. RESULTS: Serum 24,25(OH)2D3 strongly correlated with 25D3 in both black and white study participants (r = 0.90, P < 0.001 and r = 0.86, P < 0.001 respectively). Blacks had lower mean 25D3 than whites [17.0 (7.8) vs 27.5 (11.3) ng/mL; 42.4 (19.5) vs 68.6 (28.2) nmol/L, P < 0.001] and lower mean 24,25(OH)2D3 [2.1 (1.3) vs 3.6 (2.0) ng/mL; 5.1 (3.1) vs 8.7 (4.8) nmol/L, P < 0.001]. In contrast to total 25D3 concentrations, mean VMR values were similar in blacks and whites [11.9 (4.0) vs 12.5 (3.4), P = 0.16, respectively] and were negatively correlated with parathyroid hormone concentrations in both races (rs = -0.26, P < 0.001, and rs = -0.25, P < 0.001, respectively). CONCLUSIONS: Our results provide further evidence that measurement of total 25D for assessment of vitamin D status in patients of African descent deserves reevaluation and suggest that alternative measures such as VMR should be considered.

Can vitamin D metabolite measurements facilitate a "treat-to-target" paradigm to guide vitamin D supplementation?

UNLABELLED: Substantial variability exists in the serum 25(OH)D increase observed in response to vitamin D supplementation. Measurement of circulating cholecalciferol and 24,25(OH)2D, as indicators of vitamin D absorption and degradation, respectively, account for approximately half of the variation in serum 25(OH)D observed following supplementation. INTRODUCTION: Vitamin D supplementation produces a variable response in serum 25(OH)D. This variability likely reflects, in part, differences in vitamin D absorption and/or degradation. Despite this variation in response, virtually all expert recommendations endorse a fixed vitamin D supplementation dose, an approach also used in most prospective studies. Such utilization of a single vitamin D dose does not assure attaining any pre-specified target 25(OH)D level, thereby compromising clinical care and prospective supplementation trials. This study begins addressing this weakness by exploring the feasibility of vitamin D metabolite measurements to predict serum 25(OH)D level attained following supplementation. METHODS: Ninety-one community-dwelling postmenopausal women with baseline 25(OH)D of 10-30 ng/mL received oral vitamin D3, 2300 or 2500 IU, daily for 4-6 months. Serum 25(OH)D, cholecalciferol (D3), and 24,25(OH)2D were measured before and at the end of supplementation to determine if metabolite concentrations allow prediction of the 25(OH)D level attained. RESULTS: From baseline and follow-up data, we derived a multiple linear regression model predicting posttreatment 25(OH)D as follows: final 25(OH)D = 8.3 + (1.05*initial 25(OH)D) - (7.7*initial 24,25(OH)2D) + (0.53*final D3) + (4.2*final 24,25(OH)2D). This model has an adjusted R(2) = 0.55, thus accounting for approximately half of the observed variance in the final 25(OH)D level. CONCLUSIONS: The contributions of circulating cholecalciferol and 24,25(OH)2D to this predictive model can be considered as indicators of intestinal absorption and clearance, respectively. This paradigm requires further study; it may allow efficient "treat-to-25(OH)D-target" strategies useful in optimizing prospective studies and clinical practice.

[Assessment of Vitamin D Metabolism by LC-MS/MS].

Vitamin D is pro-hormone that has important roles in calcium metabolism in the intestine, kidneys, and bone. Many studies have indicated that vitamin D deficiency causes not only rickets and osteomalacia, but also increases the risk of various diseases such as cancer and autoimmune disease. Of the many vitamin D metabolites, 25-hydroxyvitamin D[25OH-D], 1α,25-dihydroxyvitamin D[1,25(OH)2-D], and 24,25-dihydroxyvitamin D [24,25 (OH) 2-D] are important for assessing vitamin D metabolism. They are circulating, biologically active, and major inactive forms of vitamin D metabolite, respectively. Immunoassays are widely used for the measurement of serum/plasma vitamin D metabolites. With such an assay, however, the accurate quantification of these metabolites is difficult because of cross-reactivity. Liquid chromatography-tandem mass spectrometry [LC-MS/MS] is the gold standard method for analyzing these metabolites due to its high sensitivity and selectivity. Derivatization with a Cookson-type reagent is a key technique for robust and sensitive analysis by LC-MS/MS. A Cookson-type reagent rapidly and quantitatively reacts with the s-cis-diene structure of vitamin D metabolites, and markedly enhances the ionization efficiency. Using a recently developed Cookson-type reagent (DAPTAD), we successfully established an LC-MS/MS-based method for analyzing serum vitamin D metabolites including 250H-D, 1,25 (OH) 2-D, and 24,25 (OH) 2-D. Thus, LC-MS/MS can be a powerful tool for the accurate determination of vitamin D metabolism. In this review, we describe the advantages of LC-MS/MS for the determination of vitamin D metabolites.

Estimated GFR and circulating 24,25-dihydroxyvitamin D3 concentration: a participant-level analysis of 5 cohort studies and clinical trials.

BACKGROUND: Decreased glomerular filtration rate (GFR) leads to reduced production of 1,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3 (25[OH]D3). Effects of low GFR on vitamin D catabolism are less well understood. We tested associations of estimated GFR (eGFR) with the circulating concentration of 24,25-dihydroxyvitamin D3 (24,25[OH]2D3), the most abundant product of 25(OH)D3 catabolism, across populations with a wide range of GFRs. STUDY DESIGN: Cross-sectional study. SETTING & PARTICIPANTS: 9,596 participants in 5 cohort studies and clinical trials: the Diabetes Control and Complications Trial (N=1,193), Multi-Ethnic Study of Atherosclerosis (N=6,470), Cardiovascular Health Study (N=932), Seattle Kidney Study (N=289), and Hemodialysis Study (N=712). PREDICTOR: eGFR. OUTCOME: Circulating 24,25(OH)2D3 concentration. MEASUREMENTS: GFR was estimated from serum creatinine using the Chronic Kidney Disease Epidemiology Collaboration equation. Vitamin D metabolites were measured by mass spectrometry. RESULTS: Circulating 24,25(OH)2D3 concentration was correlated with circulating 25(OH)D3 concentration (Pearson r range, 0.64-0.88). This correlation was weaker with lower eGFRs. Moreover, the increment in 24,25(OH)2D3 concentration associated with higher 25(OH)D3 concentration (slope) was lower with lower eGFRs: 2.06 (95% CI, 2.01-2.10), 1.77 (95% CI, 1.74-1.81), 1.55 (95% CI, 1.48-1.62), 1.17 (95% CI, 1.05-1.29), 0.92 (95% CI, 0.74-1.10), 0.61 (95% CI, 0.22-1.00), and 0.37 (95% CI, 0.35-0.39) ng/mL of 24,25(OH)2D3 per 10 ng/mL of 25(OH)D3 for eGFRs≥90, 60-89, 45-59, 30-44, 15-29, and <15 mL/min/1.73 m2 and end-stage renal disease treated with hemodialysis, respectively. As a result, at a 25(OH)D3 concentration of 20 ng/mL, mean 24,25(OH)2D3 concentrations were 2.92 (95% CI, 2.87-2.96), 2.68 (95% CI, 2.64-2.72), 2.35 (95% CI, 2.26-2.45), 1.92 (95% CI, 1.74-2.10), 1.69 (95% CI, 1.43-1.95), 1.14 (95% CI, 0.62-1.66), and 1.04 (95% CI,1.02-1.07) ng/mL for each category, respectively. This interaction was independent of other relevant clinical characteristics. Race, diabetes, urine albumin excretion, and circulating parathyroid hormone and fibroblast growth factor 23 concentrations more modestly modified the association of 24,25(OH)2D3 with 25(OH)D3. LIMITATIONS: Lack of direct pharmacokinetic measurements of vitamin D catabolism. CONCLUSIONS: Lower eGFR is associated strongly with reduced vitamin D catabolism, as measured by circulating 24,25(OH)2D3 concentration.

Dietary vitamin D inadequacy accelerates calcification and osteoblast-like cell formation in the vascular system of LDL receptor knockout and wild-type mice.

Vitamin D insufficiency is highly associated with cardiovascular morbidity and mortality. We have demonstrated enhanced vascular calcification in LDL receptor knockout (LDLR(-/-)) mice fed a diet low in vitamin D. This study aimed to investigate the impact of a diet low in vitamin D on vascular calcification in wild-type (WT) mice lacking atherosclerotic plaques and the effects of a persistent and discontinuous vitamin D insufficiency on atherosclerotic plaque composition in LDLR(-/-) mice. The study was performed with 4-wk-old male WT and LDLR(-/-) mice that were fed a normal calcium/phosphate Western diet (210 g/kg fat, 1.5 g/kg cholesterol) containing either adequate (+D; 1000 IU/kg) or low (-D; 50 IU/kg) amounts of vitamin D-3 for 16 wk. Four groups of LDLR(-/-) mice received 1 of the 2 diets for additional 16 wk (total 32 wk) and were compared with mice fed the diets for only 16 wk. WT and LDLR(-/-) mice that were fed the -D diet for 16 wk tended to develop more calcified spots in the aortic valve than mice fed the +D diet (+50% and +56%, respectively; P < 0.10). In LDLR(-/-) mice, the extent of calcification increased from week 16 to week 32 and was higher in the -D than in the +D group (P < 0.05). The calcification, owing to the -D diet, was accompanied by highly expressed osteoblast differentiation factors, indicating a transdifferentiation of vascular cells to osteoblast-like cells. Feeding the +D diet subsequent to the -D diet reduced the vascular calcification (P < 0.05). LDLR(-/-) mice fed the -D diet for 32 wk had higher plaque lipid depositions (+48%, P < 0.05) and a higher expression of cluster of differentiation 68 (+31%, P < 0.05) and tumor necrosis factor α (+134%, P < 0.001) than the +D group. Collectively, the findings imply low vitamin D status as a causal factor for vascular calcification and atherosclerosis.

Dysregulated mineral metabolism in patients with acute kidney injury and risk of adverse outcomes.

OBJECTIVE: Numerous studies have evaluated the prevalence and importance of vitamin D deficiency among patients with chronic kidney disease and end-stage renal disease; however, little is known about vitamin D levels in acute kidney injury (AKI). We evaluated the association between vitamin D metabolites and clinical outcomes among patients with AKI. DESIGN: Prospective cohort study. PATIENTS: A total of 30 participants with AKI and 30 controls from general hospital wards and intensive care units at a tertiary care hospital were recruited for the study. MEASUREMENTS: Plasma levels of 25-hydroxyvitamin D [25(OH)D], 1,25-dihydroxyvitamin D [1,25(OH)2 D], 24R,25-dihydroxyvitamin D3 , vitamin D binding protein (VDBP) and fibroblast growth factor 23 (FGF23) were measured within 24 hours of AKI onset and 5 days later. Bioavailable 25(OH)D and 1,25(OH)2 D levels, defined as the sum of free- and albumin-bound 25(OH)D and 1,25(OH)2 D, were estimated using equations. RESULTS: Compared to controls, participants with AKI had lower levels of 1,25(OH)2 D [17 (10-22) vs 25 (15-35) pg/ml, P = 0·01], lower levels of VDBP [23 (15-31) vs 29 (25-36) mg/dl, P = 0·003] and similar levels of bioavailable 25(OH)D and 1,25(OH)2 D at enrolment. Levels of bioavailable 25(OH)D were inversely associated with severity of sepsis in the overall sample (P < 0·001). Among participants with AKI, bioavailable 25(OH)D, but not other vitamin D metabolites, was significantly associated with mortality after adjusting for age and serum creatinine (adjusted odds ratio per 1 SD ln [bioavailable 25(OH)D] = 0·16, 95% confidence interval = 0·03-0·85). CONCLUSIONS: Bioavailable 25(OH)D could have a role as a biomarker or mediator of adverse outcomes among patients with established AKI.

Interdependence and contributions of sun exposure and vitamin D to MRI measures in multiple sclerosis.

PURPOSE: To assess the relationships of sun exposure history, supplementation and environmental factors to vitamin D levels in multiple sclerosis (MS) patients and to evaluate the associations between sun exposure and MRI measures. METHODS: This study included 264 MS patients (mean age 46.9±10 years, disease duration 14.6±10 years; 67.8% relapsing-remitting, 28% secondary progressive and 4.2% primary progressive MS) and 69 healthy controls. Subjects underwent neurological and 3 T MRI examinations, provided blood samples and answered questions to a structured questionnaire. Information on race, skin and eye colour, supplement use, body mass index (BMI) and sun exposure was obtained by questionnaire. The vitamin D metabolites (25-hydroxy vitamin D3, 1, 25-dihydroxy vitamin D3 and 24, 25-dihydroxy vitamin D3) were measured using mass spectrometry. RESULTS: Multivitamin supplementation (partial correlation r(p)=0.29, p<0.001), BMI (r(p)=-0.24, p=0.001), summer sun exposure (r(p)=0.22, p=0.002) and darker eye colour (r(p)=-0.18, p=0.015) had the strongest associations with vitamin D metabolite levels in the MS group. Increased summer sun exposure was associated with increased grey matter volume (GMV, r(p)=0.16, p=0.019) and whole brain volume (WBV, r(p)=0.20, p=0.004) after correcting for Extended Disability Status Scale in the MS group. Inclusion of 25-hydroxy vitamin D3 levels did not substantially affect the positive associations of sun exposure with WBV (r(p)=0.18, p=0.003) and GMV (r(p)=0.14, p=0.026) in the MS group. CONCLUSIONS: Sun exposure may have direct effects on MRI measures of neurodegeneration in MS, independently of vitamin D.

Anti-inflammatory effect of vitamin D on gingivitis: a dose-response randomised control trial.

PURPOSE: To assess the anti-inflammatory effect of vitamin D on gingivitis at various doses. MATERIALS AND METHODS: In this randomized controlled trial, daily oral vitamin D supplementation was given in doses of 2000 IU for group A, 1000 IU for group B, 500 IU for group C and a placebo for group D over a 3-month period. The changes in gingival scores were measured after the 1st, 2nd and 3rd months. RESULTS: The gingivitis score changed in direct proportion to the dose of vitamin D supplementation. In group A, the mean gingival scores were 2.4 (baseline), 1.7 after the first month, 0.8 after the second month and 0.3 after the third month. The group B mean baseline gingival score of 2.3 decreased to 2.0 in the first month, 1.1 after the second month and 0.5 after the third month. In group C, the baseline gingival scores were 2.2 and 1.9 after one month, 1.4 after two months and 0.8 by the last visit. Comparing baseline gingivitis scores with the later-visit score using the Wilcoxon paired test, the significant anti-inflammatory effect was seen in group A after one month, in group B at two months and in group C at three months after oral vitamin D supplementation (P < 0.0001). However, group D did not show a significant antiinflammatory effect. CONCLUSION: There is a dose-dependent anti-inflammatory effect of vitamin D on gingivitis. Vitamin D is a safe and effective anti-inflammatory agent in doses ranging from 500 IU to 2000 IU. Results are apparent earlier with the higher dose of 2000 IU.

Vitamin D metabolites are associated with clinical and MRI outcomes in multiple sclerosis patients.

PURPOSE: The associations between vitamin D and MRI measures of brain tissue injury have not been previously investigated in multiple sclerosis (MS). This research evaluates the significance of vitamin D and its active metabolites in brain tissue injury and clinical disability in MS patients. METHODS: The study population consisted of 193 MS patients (152 women and 41 men; mean age 46.1 (SD 8.4) years; disease duration 13.8 (SD 8.4) years). Serum levels of 25-hydroxyvitamin D(3) (25(OH)VD(3)), 25-hydroxyvitamin D(2) (25(OH)VD(2)), 1α, 25-dihydroxyvitamin D(3) (1, 25(OH)(2)VD(3)) and 24(R), 25-dihydroxyvitamin D(3) (24, 25(OH)(2)VD(3)) were measured using a novel capillary liquid-chromatography-mass spectrometry method. Disability was assessed with the Expanded Disability Status Scale (EDSS) and the MS Severity Scale (MSSS). MRI measures included T2 lesion volume (LV), T1-LV and brain parenchymal fraction. The associations between deseasonalised levels of vitamin D metabolites and clinical and MRI measurements were assessed using regression analyses. RESULTS: Lower deseasonalised levels of total 25(OH)VD (p=0.029), 25(OH)VD(3) (p=0.032) and 24, 25(OH)(2)VD(3) (p=0.005) were associated with higher MSSS. Similarly, lower deseasonalised levels of 24, 25(OH)(2)VD(3) (p=0.012) were associated with higher EDSS. Higher values of the 25(OH)VD(3) to 24, 25(OH)(2)VD(3) ratio were associated with higher MSSS (p=0.041) and lower brain parenchymal fraction (p=0.008). CONCLUSIONS: Vitamin D metabolites have protective associations with disability and brain atrophy in MS. In particular, the results indicate strong associations for the 24, 25(OH)(2)VD(3) metabolite, which has not been extensively investigated in MS patients.