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Background/aims: Fibroblast growth factor (FGF)-23 and parathyroid hormone (PTH) are both potent phosphaturic hormones. Since they exert opposite effects on vitamin D metabolism, the measurement of 3 vitamin D metabolites; 25-hydroxyvitamin D (25-OH-D), 1,25-dihydroxyvitamin D (1,25(OH)2D), and 24,25-dihydroxyvitamin D (24,25(OH)2D), allows the distinction of the effects of FGF-23 from those of PTH. The aim of this study was to elucidate which factor, FGF-23 or PTH, plays a more important role in the regulation of vitamin D metabolites in subjects with estimated glomerular filtration (eGFR) ≥60 ml/min/1.73 m(2)., Methods: Subjects with eGFR ≥60 ml/min/1.73 m(2) (n = 20) were enrolled and their serum levels of FGF-23, intact PTH, and vitamin D metabolites were determined., Results: Serum FGF-23 correlated inversely with 1,25(OH)2D (r = -0.717, p = 0.0004) and the 1,25(OH)2D/25-OH-D ratio (r = -0.518, p = 0.019), compared with a significant positive correlation between serum intact PTH and the 1,25(OH)2D/25-OH-D ratio (r = 0.562, p = 0.010). Multiple regression analyses revealed serum FGF-23 as a significant factor that was associated with serum 1,25(OH)2D (β = -0.593, p = 0.018), 1,25(OH)2D/25-OH-D ratio (β = -0.521, p = 0.025), and the 24,25(OH)2D/1,25(OH)2D ratio (β = 0.632, p = 0.008), and intact PTH as a significant factor associated with the 1,25(OH)2D/25-OH-D ratio (β = 0.445, p = 0.028)., Conclusions: This study demonstrated that, even in subjects with eGFR ≥60 ml/min/1.73 m(2), FGF-23 might play an important role in the regulation of vitamin D metabolism. In addition to the established role of PTH, the association between FGF-23 and indices of vitamin D metabolism suggested the potential role of FGF-23 on phosphate metabolism in such patients., (© 2015 S. Karger AG, Basel.)

Background/objectives: Based on the growing evidence of risk reduction from fresh fruit and vegetable consumption and an inverse relationship between serum 25-hydroxyvitamin D (25OHD) and the risk of type 2 diabetes (T2D), we determined the benefits of regularly consuming vitamin D-enriched mushrooms in a prediabetic cohort. Exposing edible mushrooms to ultraviolet B (UVB) light increases vitamin D2 (D2) and raises serum 25OHD2 in healthy young adults; however, their benefit to deficient prediabetics and glucose metabolism remains untested., Subjects/methods: Forty-three prediabetic, D-deficient adults (25OHD≤20 ng/ml), BMI>25 were randomized to four groups consuming daily entrées containing 100 g fresh sliced cooked mushrooms prepared by a chef for 16 weeks. Two groups were fed UVB-treated mushrooms initially containing: 600 IU D2 or 4000 IU D2; each one also received one capsule of placebo daily. Two control groups were fed untreated mushrooms and D3 dietary supplements at two label doses: 600 IU D3 and 4000 IU D3. D2 and D3 content were analyzed in mushrooms, before and after cooking and in over-the-counter supplements., Results: After 16 weeks, both D2-UVB-mushroom entrée doses, which were significantly lower after cooking, produced modest or no increases in 25OHD2 or total 25OHD relative to the positive control subjects who actually consumed about 1242 and 7320 IU per day of D3 (higher than stated on the label)., Conclusions: Unanticipated D2 cooking loss from fresh UVB mushrooms and probable low absorption and/or hydroxylation may explain the smaller increase in 25OHD2 in our prediabetic overweight/obese cohort compared with past findings in younger, healthy subjects. Moreover, no dose or vitamin D source was associated with modifying T2D risk factors.

Importance for Vit D estimation has increased in the recent years due to its link to various diseases. Measurements of Vit D by different diagnostic laboratories is however not uniform. There is variation pertaining to assay methodology and also variation in the measurement of different metabolites of Vit D. There are also various confounders which influence Vit D assays and which in most instances are overlooked. Also a matter of concern regarding Vit D assays is the lack of assay standardisation. These factors contribute to the variation in the reports generated by the diagnostic laboratories. Therefore interpretation of Vit D reports needs proper understanding of these interfering factors and further reports need to be correlated substantially with the clinical findings.

Background: Interactions between calcium and vitamin D may have implications for the regulation of serum 25-hydroxyvitamin D [25(OH)D] and its catabolism and, consequently, the vitamin D dietary requirement., Objective: We investigated whether different calcium intakes influenced serum 25(OH)D and indexes of vitamin D activation and catabolism during winter and in the context of both adequate and inadequate vitamin D intakes., Design: A 15-wk winter-based, randomized, placebo-controlled, double-blind vitamin D₃ intervention (20 μg/d) study was carried out in free-living men and women aged ≥50 y (n = 125) who were stratified according to calcium intakes [moderate-low (<700 mg/d) or high (>1000 mg/d) intake]. The serum 25(OH)D concentration was the primary outcome, and serum calcium, parathyroid hormone (PTH), 1,25-dihydroxyvitamin D [1,25(OH)₂D], 24,25-dihydroxyvitamin D [24,25(OH)₂D], the ratio of 24,25(OH)₂D to 25(OH)D, vitamin D-binding protein, and free 25(OH)D were exploratory outcomes., Results: A repeated-measures ANOVA showed there was no significant (P = 0.2) time × vitamin D treatment × calcium intake grouping interaction effect on the mean serum 25(OH)D concentration over the 15-wk intervention period. Serum 25(OH)D concentrations increased (P ≤ 0.005) and decreased (P ≤ 0.002) in vitamin D₃ and placebo groups, respectively, and were of similar magnitudes in subjects with calcium intakes <700 mg/d (and even <550 mg/d) compared with >1000 mg/d. The response of serum PTH, 1,25(OH)₂D, 24,25(OH)₂D, the ratio of 24,25(OH)₂D to 25(OH)D, and free 25(OH)D significantly differed in vitamin D₃ and placebo groups but not by calcium intake grouping., Conclusions: We found no evidence of a vitamin D sparing effect of high calcium intake, which has been referred to by some authors as "vitamin D economy." Thus, recent dietary vitamin D requirement estimates will cover the vitamin D needs of even those individuals who have inadequate calcium intakes., (© 2014 American Society for Nutrition.)

Objective: To investigate seasonal variation of vitamin D levels in 148,821 serum samples during a 2year time period in a northern-latitude city in the United States., Methods: Total vitamin D assay testing by chemiluminescence was performed on the DiaSorin Liaison. Vitamin D results were extracted from the laboratory information system without patient identification during 2011 and 2012 and separated by season and vitamin D results: less than 10ng/mL (deficient), 10-20.0ng/mL (insufficient), 20.1-30ng/mL (borderline), 30.1-40ng/mL (sufficient), 40.1-100ng/mL, and greater than 100ng/mL., Results: The seasonal winter period constituted the months of January through March; spring, April through June; summer, July through September; and fall, October through December. The data set analyzed included 36,643 samples during the winter, 38,299 in spring, 36,141 in summer, and 37,738 in fall and demonstrated an expected rise and fall in vitamin D levels., Conclusion: This retrospective epidemiological study demonstrates seasonal variation of vitamin D levels at clinical decision points. Although not unexpected, this variation has an impact on studies relating low vitamin D levels to higher rates of cancer, cardiovascular disease, multiple sclerosis, diabetes, autoimmune disease, and a host of other health risk assessments., (Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Context: We report the presence of substantial concentrations of 3-epi-25(OH)D3 in two patients and a third patient with 3-epi-25(OH)D2., Patients: The first patient, a 66-year-old female receiving cholecalciferol 4000 IU daily was originally found to have 53 ng/mL of 25(OH)D3 and almost an equal amount of 3-epi-25(OH)D3. Subsequently, the patient had four additional samples, each of which has similar levels of both 25(OH)D3 and 3-epi-25(OH)D3. The second patient, a 7-year-old male receiving cholecalciferol 1000 IU daily, had a 25(OH)D3 concentration of 37 ng/mL and 3-epi-25(OH)D3 of approximately 10 ng/mL. The third and most intriguing patient, a 55-year-old female was receiving ergocalciferol 50,000 IU twice weekly for approximately 3 months, at which time her serum 25(OH)D2 was 64 ng/mL and her 3-epi-25(OH)D2 was approximately 32 ng/mL. This patient's physician changed her vitamin D therapy to cholecalciferol 1000 IU daily, discontinuing ergocalciferol, and a second specimen was collected 5 months later. Analysis of this last specimen found both 25(OH)D3 and 25(OH)D2 at concentrations of 12 and 24 ng/mL respectively, plus corresponding 3-epimer peaks for both 25(OH)D3 and 25(OH)D2 observed chromatographically., Conclusion: The presence of a substantial concentration of 3-epi-25(OH)D in these three patients documents that one cannot assume 3-epi is a trivial metabolite of 25(OH)D for all patients. In addition, the appearance of 3-epi-25(OH)D3 when the last patient changed her vitamin D supplementation from ergocalciferol to cholecalciferol demonstrates that the 3-epimer is probably an endogenous metabolite of 25(OH)D in humans.

Active vitamin D metabolites 1,25-dihydroxyvitamin D2 [1,25-(OH)2-D2; derived from ergocalciferol] and D3 [1,25-(OH)2-D3; derived from cholecalciferol] are found in low levels in the circulation and require a very sensitive method for measurement. Radioimmunoassay (RIA) has been the method of choice, but it lacks the specificity needed to distinguish between 1,25-(OH)2-D2 and -D3, whereas liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods have the advantage of high specificity and sensitivity. Here, we compare a new derivative for ionizing 1,25-(OH)2-D to enhance the signal and provide the most sensitive assay for measuring vitamin D. We used the Amplifex diene method of derivatizing prior to LC/MS/MS and compared it to the standard RIA method and the 4-phenyl-1,2,4-triazole-3,5-dione (PTAD) method of derivatizing prior to LC/MS/MS. In the evaluation of 20 human serum samples, all methods correlated strongly across the upper levels of the standard 1,25-(OH)2-D2 and -D3 ranges (Amplifex and RIA, pc=0.97; Amplifex and PTAD, pc=0.96) but less strongly on the lower levels of the standard range (Amplifex and RIA, pc=0.81; Amplifex and PTAD, pc=0.65) suggesting differences in the sensitivities between the assays. The Amplifex method was determined to be more sensitive than the PTAD method, as peak areas were significantly higher for the Amplifex method and provided for a 10 fold higher signal-to-noise ratio than PTAD. Therefore, the Amplifex LC/MS/MS method is the most sensitive and specific method available for measuring 1,25-(OH)2-D2 and -D3 while using the smallest sample volume., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Scope: Poor vitamin D (vitD) status is linked to increased risk of infectious diseases, thus there is need for vitD-rich foods. UVB-exposed mushrooms synthesize vitD₂ but knowledge of bioavailability and function in immune response is lacking., Methods and Results: One hundred rats were fed one of five diets--control, 20 IU vitD₃/day; no vitD₃/day; 5% unexposed mushroom, 2.4 IU vitD₂/day; 2.5% UVB mushroom, 300 IU vitD₂/day; and 5% UVB mushroom, 600 IU vitD₂/day--for 10 wk and challenged with either saline or the endotoxin LPS. Blood and tissues were collected at 3 h postchallenge. Plasma 25-hydroxyvitamin D (25OHD) levels from UVB-exposed mushroom fed rats were significantly elevated and associated with higher natural killer cell activity and reduced plasma inflammatory response to LPS compared to control diet fed rats. Microarray evaluation of rat spleens for changes in inflammatory gene expression showed significant upregulation of proinflammatory genes after LPS compared to saline controls in all groups. However, compared to control rats, upregulation of the proinflammatory genes was markedly reduced in the groups fed vitD₂-enriched mushrooms., Conclusion: Rats fed UVB-exposed mushrooms had significantly higher plasma total 25OHD levels that were associated with increased innate immune response and anti-inflammatory effects., (Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. This article is a U.S. Government work and is in the public domain in the USA.)

Background: Vegan and vegetarian diets could overcome many diseases of civilization. This study examines whether a whole food vegan diet with Nori algae and wild mushrooms can provide a sufficient quantity of critical nutrients., Methods: Five blood samples (Baseline to Time 5) were taken over eight months from 75 subjects (10 vegans without B12 supplementation who consumed Nori algae and wild mushrooms, 20 vegans with supplementation, 40 vegetarians, 5 meat-eaters). Blood was analyzed for blood cell counts, total vitamin B12, holotranscobalamin, homocysteine, methylmalonic acid, vitamin B6, folic acid, ferritin, TSH, zinc, creatinine, vitamin D2 and D3., Results: In the vegan group without supplementation, all means were within the tolerance (holotranscobalamin, homocystein) or normal, except for elevated methylmalonic acid and diminished vitamin D. This group developed significantly higher vitamin D2 levels. The vegan group with B12 supplementation and the vegetarian group showed normal values for all parameters., Conclusions: Vegans following a whole food diet had a borderline supply of vitamin B12. Folic acid, vitamin B6, TSH, iron metabolism, and the blood count were in the normal range. Vegans taking dietary supplements demonstrated satisfactory overall results. An ingestion of sundried mushrooms can contribute to the supply of vitamin D.

This study determined if 6-weeks vitamin D2 supplementation (vitD2, 3800 IU/day) had an influence on muscle function, eccentric exercise-induced muscle damage (EIMD), and delayed onset of muscle soreness (DOMS) in National Association for Stock Car Auto Racing (NASCAR) NASCAR pit crew athletes. Subjects were randomized to vitD2 (n=13) and placebo (n=15), and ingested supplements (double-blind) for six weeks. Blood samples were collected and muscle function tests conducted pre- and post-study (leg-back and hand grip dynamometer strength tests, body weight bench press to exhaustion, vertical jump, 30-s Wingate test). Post-study, subjects engaged in 90 min eccentric-based exercise, with blood samples and DOMS ratings obtained immediately after and 1- and 2-days post-exercise. Six weeks vitD2 increased serum 25(OH)D2 456% and decreased 25(OH)D3 21% versus placebo (p<0.001, p=0.036, respectively), with no influence on muscle function test scores. The post-study eccentric exercise bout induced EIMD and DOMS, with higher muscle damage biomarkers measured in vitD2 compared to placebo (myoglobin 252%, 122% increase, respectively, p=0.001; creatine phosphokinase 24 h post-exercise, 169%, 32%, p<0.001), with no differences for DOMS. In summary, 6-weeks vitD2 (3800 IU/day) significantly increased 25(OH)D2 and decreased 25(OH)D3, had no effect on muscle function tests, and amplified muscle damage markers in NASCAR pit crew athletes following eccentric exercise.

Context: There is no doubt that vitamin D must be activated to the hormonal form 1,25-dihydroxyvitamin D to achieve full biological activity or that many tissues participate in this activation process-be it endocrine or autocrine. We believe that not only is 25-hydroxyvitamin D important to tissue delivery for this activation process, but also that intact vitamin D has a pivotal role in this process., Objective: In this review, evidence on the vitamin D endocrine/autocrine system is presented and discussed in relation to vitamin D-binding protein affinity, circulating half-lives, and enzymatic transformations of vitamin D metabolites, and how these affect biological action in any given tissue., Conclusions: Circulating vitamin D, the parent compound, likely plays an important physiological role with respect to the vitamin D endocrine/autocrine system, as a substrate in many tissues, not originally thought to be important. Based on emerging data from the laboratory, clinical trials, and data on circulating 25-hydroxyvitamin D amassed during many decades, it is likely that for the optimal functioning of these systems, significant vitamin D should be available on a daily basis to ensure stable circulating concentrations, implying that variation in vitamin D dosing schedules could have profound effects on the outcomes of clinical trials because of the short circulating half-life of intact vitamin D.

Background: Estrogen therapy (ET) is associated with lower serum calcium and phosphorus concentrations and is known to increase bone mineral density (BMD). Other biomarkers of mineral metabolism may help understand the biological basis of these actions., Methods: We studied 2767 postmenopausal women in the Multi-Ethnic Study of Atherosclerosis, 862 (31%) of whom were using ET. We measured serum concentrations of calcium, phosphorus, 25-hydroxyvitamin D, 24,25-dihydoxyvitamin D, and fibroblast growth factor-23 and urinary fractional excretion of calcium (FEca) and phosphorus (FEphos). We examined the associations of ET with each biomarker. In addition, we tested whether the adjustment for biomarkers attenuated the association of ET with lumbar BMD measured by abdominal computed tomography in a subset of 810 women., Results: In adjusted models, women who used ET were younger in age [62 (SD 8) vs 66 (9) y, P < .001], had lower mean serum calcium [-13 mg/dL (95% confidence interval [CI] -0.17, -0.10), P < .001] and lower FEca [-0.15% (95% CI -0.21, -0.09), P < .001]. Mean serum phosphorus was lower [-0.19 mg/dL (95% CI -0.23, -0.15), P < .001] and FEphos [0.56% (95% CI 0.16, 0.96), P = .007] was higher in women on ET. Mean 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D were higher [1.52 ng/dL (95% CI 0.57, 2.47), P = .002, and 0.26 ng/mL (95% CI 0.03, 0.48), P = .03, respectively] in women who used ET. Mean PTH and fibroblast growth factor-23 did not differ significantly by the use of ET. ET use was strongly associated with higher lumbar BMD [12.75 mg/cm³ (95% CI 7.77-17.73), P < .001]; however, mineral metabolism measures did not meaningfully alter this association., Conclusions: In a multiethnic cohort of postmenopausal women, ET use was associated with lower serum calcium, lower FEca, lower serum phosphorus, and higher FEphos, suggesting these associations are attributable to increased calcium intake into bone and increased urinary phosphorus excretion. ET use was also associated with greater concentrations of vitamin D metabolites. ET-associated differences in these mineral metabolism measures did not meaningfully attenuate the strong association between ET use and lumbar BMD.

Background: The bioequivalence of the different forms of vitamin D, ergocalciferol (vitamin D2) and cholecalciferol (vitamin D3), has been questioned. Earlier studies have suggested that vitamin D2 is less biologically active than vitamin D3., Objective and Design: In a parallel study, we tested the effects of supplementation with 50-μg/d doses of vitamin D2 or D3 or a placebo over a period of 8 weeks on 25(OH)D2, 25(OH)D3, their sum 25(OH)D (primary outcome variables), and PTH in healthy volunteers applying a double-blind, randomized study design. The study was conducted during the winter of 2012 in Halle (Saale), Germany, at latitude 51°47N, when UVB irradiation is virtually absent. Blood samples for the determinations of vitamin D status and PTH were collected at baseline and after 4 and 8 weeks of supplementation., Results: In the placebo group (n = 19), 25(OH)D3 decreased from 39.4 ± 14.2 to 31.1 ± 12.4 nmol/L after 8 weeks (P < .01). In the vitamin D3 group (n = 42), the concentrations of 25(OH)D3 increased from 41.5 ± 22.8 nmol/L at baseline to 88.0 ± 22.1 nmol/L after 8 weeks (P < .01). In the group receiving vitamin D2 (n = 46), the 25(OH)D2 concentrations increased significantly, whereas the 25(OH)D3 concentration fell from 36.4 ± 13.3 nmol/L at baseline to 16.6 ± 6.3 nmol/L after 8 weeks (P < .01). The total 25(OH)D was not different between the groups at baseline but differed significantly between the groups after 4 and 8 weeks (P < .001)., Conclusions: Vitamin D3 increases the total 25(OH)D concentration more than vitamin D2. Vitamin D2 supplementation was associated with a decrease in 25(OH)D3, which can explain the different effect on total 25(OH)D.

Background: The global prevalence of type 2 diabetes is increasing. Effective strategies to address this public health challenge are currently lacking. A number of epidemiological studies have reported associations between low concentrations of 25-hydroxy vitamin D and the incidence of diabetes, but a causal link has not been established. We investigate the effect of vitamin D supplementation on the metabolic status of individuals at increased risk of developing type 2 diabetes., Methods/design: In a randomised double-blind placebo-controlled trial individuals identified as having a high risk of type 2 diabetes (non-diabetic hyperglycaemia or positive diabetes risk score) are randomised into one of three groups and given 4 doses of either placebo, or 100,000 IU Vitamin D₂ (ergocalciferol) or 100,000 IU Vitamin D₃ (cholecalciferol) at monthly intervals. The primary outcome measure is the change in glycated haemoglobin level between baseline and 4 months. Secondary outcome measures include blood pressure, lipid levels, apolipoproteins, highly sensitive C-reactive protein, parathyroid hormone (PTH) and safety of supplementation. and C-reactive protein. The trial is being conducted at two sites (London and Cambridge, U.K.) and a total of 342 participants are being recruited., Discussion: Trial data examining whether supplementation of vitamin D improves glycaemic status and other metabolic parameters in people at risk of developing type 2 diabetes are sparse. This trial will evaluate the causal role of vitamin D in hyperglycaemia and risk of type 2 diabetes. Specific features of this trial include recruitment of participants from different ethnic groups, investigation of the relative effectiveness and safety of vitamin D₂ and D₃ and an evidence based approach to determination of the dose of supplementation., Trial Registration: EudraCT2009-011264-11; ISRCTN86515510.

Background: The study was conducted to evaluate the technical and clinical performance of the VITROS® Immunodiagnostic Products 25-OH Vitamin D Total Assay, and compare it with the performance of five marketed automated assays and a liquid chromatography/mass spectrometry reference method (LC-MS/MS)., Methods: Three hundred patient serum samples were used to compare the correlation of the VITROS® 25-OH Vitamin D Total Assay with both the other immunoassays and the LC-MS/MS method, using Passing-Bablok regression and Bland-Altman analyses. Concordance of the diagnosis of vitamin D status was calculated to test the agreement between the different assays. In addition, samples containing vitamin D2 were used to test the assay's ability to detect the D2 form of the vitamin., Results and Conclusions: These results from the VITROS® 25-OH Vitamin D Total Assay generally correlated well with those from most of the marketed immunoassays. Cross-reactivity of the D2 form was calculated as being close to 100%. Additionally, we found substantial variability in performance amongst the various assays, which suggests the need for optimisation and recalibration of commercial methods.

Objectives: To develop a simple and sensitive LC-MS/MS procedure for quantification of serum 25-OH-vitamin D₃ (25-OH-D₃), 25-OH-vitamin D₂ (25-OH-D₂), and their C3-epimers., Methods: Serum 25-OH-vitamin D metabolites were extracted with MTBE and quantified by LC-MS/MS. Commercially available calibrators and QC materials were employed. The ion-transition 401.2→365.2 was monitored for 25-OH-D₃ and C3-epi-25-OH-D₃, 407.2→371.3 for d6-25-OH-D₃, 413.2→331.2 for 25-OH-D₂ and C3-epi-25-OH-D₂ and 419.2→337.1 for, d6-25-OH-D₂. As a proof-of-principle, 25-OH-D₃ and C3-epi-25-OH-D₃ were quantified in 200 pediatric subjects (0-20 years of age). Cholecalciferol supplements were examined as a potential source of C3-epimer., Results: The assay provided an LLOQ of ≤2.8 nmol/L for all 25-OH-D metabolites, with a linear response up to 400 nmol/L. The CV was <10% for 25-OH-D₂/₃ and <15% for C3-epi-25-OH-D₃. C3-epi-25-OH-D₃ was quantified in all subjects, with higher concentrations observed in infants ≤1 year of age (11.44 nmol/L vs. 4.4 nmol/L; p<0.001). Within the first year of life, 25-OH-D₃ concentrations increased linearly, while C3-epi-25-OH-D₃ concentrations remained constant. At 12 months of age, C3-epi-25-OH-D₃ concentration dropped by almost 50% (11.4 nmol/L in infants ≤1year of age vs. 5.4 nmol/L in infants 1-2years of age; p<0.001). Liquid vitamin D₃ supplements did not contain appreciable amounts of C3-epi-D₃., Conclusions: The proposed LC-MS/MS procedure is suitable for quantifying 25-OH-D₃ metabolites. Although the C3-epimer is present in all pediatric subjects, it is significantly elevated in individuals ≤1 year of age and drops at 12 months of age. Oral vitamin D supplements are unlikely to be a significant source of C3-vitamin D epimer., (Copyright © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Context: We previously showed that a single high dose of oral (po) cholecalciferol (D₃) sharply increases serum 25-hydroxyvitamin D [25(OH)D]., Objective: We evaluated the long-term bioavailability and metabolism of a single po or intramuscular (im) high dose of ergocalciferol (D₂) or D₃., Design: This was a prospective intervention study., Setting: The study was conducted in an ambulatory care setting., Patients: Participants were 24 subjects with hypovitaminosis D., Interventions: A single dose of 600,000 IU of po or im D₂ or D₃ was administered., Main Outcome Measures: Serum 25(OH)D and 1,25-dihydroxyvitamin D [1,25(OH)₂D] were measured at baseline and at days 30, 60, 90, and 120 by RIA. Serum 1,25(OH)₂D₂, 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], 24,25-hydroxyvitamin D₂ [24,25(OH)D₂], and 24,25-hydroxyvitamin D₃ [24,25(OH)D₃] were measured by liquid chromatography-tandem mass spectrometry in a subgroup of patients receiving the po formulations., Results: The areas under the curve of 25(OH)D after D₃ were significantly higher than those after D₂ (P < .0001). Serum 25(OH)D basal difference significantly increased at day 30 with po D₂ and D₃ (P < .01 and P < .0001) and up to day 90 with po D₃ (P < .01). The im formulations produced a slow increased, and values peaked at day 120 relative to the other time points (P < .0001). We found a decrease in 1,25(OH)₂D at day 30 (P < .05) and up to day 120 (P < .001) and an increase in 1,25(OH)₂D₂ at day 30 (P < .01) and up to day 120 (P < .01) after po D₂. Oral D₂ and D₃ produced increases in 24,25(OH)D₂ and 24,25(OH)D₃, respectively, at day 30 (P < .001)., Conclusions: A po dose of 600,000 IU of D₂ or D₃ is initially more effective in increasing serum 25(OH)D than the equivalent im dose and is rapidly metabolized. Our RIA assay for 1,25(OH)₂D may not recognize 1,25(OH)₂D₂.

The current study compared the effects of milk, yogurt or whey on the bone strength, body composition and serum biomarkers. Forty 12-week-old female Sprague-Dawley rats were ovariectomized (OVX), and another nine rats received a sham operation (Sham-Cont). After a 1-week recovery period, the OVX rats were divided into four dietary groups: OVX-control group (OVX-Cont), 17% skimmed milk powder diet group (OVX-Milk), 17% powdered fermented milk diet group (OVX-Yogurt) and 12% whey powder and 6% whey protein extract diet group (OVX-Whey) (n=10 in each group). The protein, nitrogen, fat, calcium and phosphorus contents of the experimental diets were adjusted to be similar to the control diet (AIN-93M). Eighty-four days after the beginning of the experimental diet, the total bone mineral density and bone mineral contents of lumbar vertebrae were significantly higher in the OVX-Milk and OVX-Whey groups than in the OVX-Cont group. Furthermore, the level of 1alpha, 25-dihydroxyvitamin D3 [1alpha, 25(OH)2D3] was significantly lower, while the serum level of FGF23 was significantly higher in the OVX-Milk, OVX-Yogurt and OVX-Whey groups than in the OVX-Cont group. These findings suggest that milk and the dairy products could improve bone metabolism in a postmenopausal animal model at least partly through changing the balance between 1alpha, 25(OH)2D3 and FGF23., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Context: The relationships among cortical volumetric bone mineral density (CortBMD) and comprehensive measures of mineral metabolism have not been addressed in chronic kidney disease (CKD)., Objective: The aim of the study was to identify the determinants of CortBMD in childhood CKD. A secondary objective was to assess whether CortBMD was associated with subsequent fracture., Design and Participants: This prospective cohort study included 171 children, adolescents, and young adults (aged 5-21 years) with CKD stages 2-5D at enrollment and 89 1 year later., Outcomes: Serum measures included vitamin D [25-hydroxyvitamin D (25[OH]D), 1,25-dihydroxyvitamin D (1,25(OH)₂D), 24,25-dihydroxyvitamin D], vitamin D-binding protein, intact PTH, fibroblast growth factor 23, calcium, and phosphorus. Tibia quantitative computed tomography measures of CortBMD were expressed as sex-, race-, and age-specific Z-scores based on 675 controls. Multivariable linear regression identified the independent correlates of CortBMD Z-scores and the change in CortBMD Z-scores., Results: Lower calcium (β = .31/1 mg/dL, P = .01) and 25(OH)D (β = .18/10 ng/mL, P = .04) and higher PTH (β = -.02/10%, P = .002) and 1,25(OH)₂D (β = -.07/10%, P < .001) were independently associated with lower CortBMD Z-scores at baseline. The correlations of total, free, and bioavailable 25(OH)D with CortBMD did not differ. Higher baseline 1,25(OH)₂D (P < .05) and greater increases in PTH (P < .001) were associated with greater declines in CortBMD Z-scores. Greater increases in calcium concentrations were associated with greater increases in CortBMD Z-scores in growing children (interaction P = .009). The hazard ratio for fracture was 1.75 (95% confidence interval 1.15-2.67; P = .009) per SD lower baseline CortBMD., Conclusions: Greater PTH and 1,25(OH)₂D and lower calcium concentrations were independently associated with baseline and progressive cortical deficits in childhood CKD. Lower CortBMD Z-score was associated with increased fracture risk.

Background: It is not known whether genetic variation in the vitamin D binding protein (DBP) influences 25-hydroxyvitamin D levels [25(OH)D] after vitamin D supplementation. We aimed to investigate the changes of total 25(OH)D, 25(OH)D₃ and 25(OH)D₂ in a Thai cohort, according to type of vitamin D supplement (vitamin D₃ or D₂) and DBP genotype, after receiving vitamin D₃ or D₂ for 3 months., Methods: Thirty-nine healthy subjects completed the study. All subjects received 400  IU of either vitamin D₃ or D₂, plus a calcium supplement, every day for 3 months. Total serum 25(OH)D, 25(OH)D₃ and 25(OH)D₂ were measured by LC-MS/MS. Individual genotyping of rs4588 in the DBP gene was performed using real-time PCR., Results: Vitamin D₃ supplementation of 400  IU/d increased 25(OH)D₃ significantly (+16.2 ± 4.2 nmol/L, p <0.001). Vitamin D₂ (400 IU/d) caused increased 25(OH)D₂ levels (+22.0 ± 2.11 nmol/L, p <0.001), together with a decrease of 25(OH)D₃ (-14.2 ± 2.0 nmol/L, p <0.001). At 3 month, subjects in vitamin D3 group tended to have higher total 25(OH)D levels than those in vitamin D₂ (67.8 ± 3.9 vs. 61.0 ± 3.0 nmol/L; p = 0.08). Subjects were then classified into two subgroups: homozygous for the DBP rs4588 C allele (CC), and the rest (CA or AA). With D₃ supplementation, subjects with CA or AA alleles had significantly less increase in 25(OH)D₃ and total 25(OH)D when compared with those with the CC allele. However, no difference was found when the supplement was vitamin D₂., Conclusion: Genetic variation in DBP (rs4588 SNP) influences responsiveness to vitamin D₃ but not vitamin D₂.

This study compared two methods of assaying the 25-hydroxylated metabolites of cholecalciferol (vitamin D3) and ergocalciferol (vitamin D2). A fully automated electrochemiluminescence assay from Roche Diagnostics and an HPLC based method from Chromsystems were used to measure vitamin D levels in surplus sera from 96 individuals, where the majority has the D2 form of the vitamin. Deming regression, concordance rate, correlation and Altman Bland agreement were performed. Seventy two subjects (75%) had a D2 concentration >10 nmol/L while the remaining twenty four subjects had vitamin D2 concentration of less than 10 nmol/L by HPLC. Overall, the Roche Diagnostics method showed a negative bias of -2.59 ± 4.11 nmol/L on the e602 as compared to the HPLC with a concordance rate of 84%. The concordance rate was 91% in samples with D2 of less than 10 nmol/L and 82% in those with D2 concentration >10 nmol/L. The overall correlation had an r value of 0.77. The r value was higher in samples with D2 levels of less than 10 nmol/L, r = 0.96, as compared to those with D2 values of greater than 10 nmol/L, r = 0.74. The observed bias had little impact on clinical decision and therefore is clinically acceptable.

Objective: The purpose of this study was to determine 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and 1,25-dihydroxyvitamin D2 [1,25(OH)2D2] levels in healthy adults consuming 1000 IU vitamin D2 or vitamin D3 per day for 11 weeks., Subjects and Design: Blood from 34 healthy male and female adults, aged 18 to 79 years, from a placebo-controlled, double-blind study who received a placebo, 1000 IU vitamin D3, or 1000 IU vitamin D2 daily for 11 weeks at end of winter was analyzed. Serum levels of 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 1,25(OH)2D2, and 1,25(OH)2D3 were determined by liquid chromatography-tandem mass spectroscopy., Results: Of the adults, 82% were vitamin D insufficient (serum 25-hydroxyvitamin D [25(OH)D <30 ng/mL]) at the start of the study. Administration of vitamin D2 and vitamin D3 induced similar increases in total 25(OH)D as well as in 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, respectively. Compared with placebo and adjusting for baseline levels, 1000 IU daily of vitamin D2 was associated with a mean increase of 7.4 pg/mL (95% confidence interval, 4.4-10.3) in 1,25(OH)2D2, which was accompanied by a mean decrease of 9.9 pg/mL (-15.8 to -4.0) in 1,25(OH)2D3. No such differences accompanied administration of 1000 IU daily of vitamin D3., Conclusion: Vitamin D2 and vitamin D3 were effective in raising and maintaining total serum concentrations of 25(OH)D. Ingestion of vitamin D2 also resulted in an increase in serum concentrations of 1,25(OH)2D2. This increase was accompanied by a comparable decrease in serum concentrations of 1,25(OH)2D3; therefore, the total 1,25-dihydroxyvitamin D [1,25(OH)2D] concentrations did not significantly change after 11 weeks compared with baseline levels. Ingestion of vitamin D3 did not alter serum concentrations of 1,25(OH)2D3 or total 1,25(OH)2D. Therefore, ingestion of 1000 IU vitamin D2 or vitamin D3 for 11 weeks was effective in raising total serum concentrations of 25(OH)D as well as sustaining serum concentrations of total 1,25(OH)2D.

Aim: Evaluation of serum ferritin and vitamin D levels in females with chronic telogen effluvium (TE) or female pattern hair loss (FPHL), in order to validate their role in these common hair loss diseases., Methods: Eighty females (18 to 45 years old) with hair loss, in the form of TE or FPHL, and 40 age-matched females with no hair loss were included in the study. Diagnosis was based upon clinical examination as well as trichogram and dermoscopy. Serum ferritin and vitamin D2 levels were determined for each participant., Results: Serum ferritin levels in the TE (14.7 ± 22.1 μg/l) and FPHL (23.9 ± 38.5 μg/l) candidates were significantly lower than in controls (43.5 ± 20.4 μg/l). Serum vitamin D2 levels in females with TE (28.8 ± 10.5 nmol/l) and FPHL (29.1 ± 8.5 nmol/l) were significantly lower than in controls (118.2 ± 68.1 nmol/l; p < 0.001). These levels decreased with increased disease severity. Serum ferritin cut-off values for TE and FPHL were 27.5 and 29.4 μg/l, respectively, and those for vitamin D were 40.9 and 67.9 nmol/l., Conclusion: Low serum ferritin and vitamin D2 are associated with hair loss in females with TE and FPHL. Screening to establish these levels in cases of hair loss and supplementing with them when they are deficient may be beneficial in the treatment of disease., (Copyright © 2013 S. Karger AG, Basel.)

Background and Objective: Low vitamin D levels correlate with measures of insulin resistance and prevalence of diabetes mellitus, but there are limited and conflicting data on changes in insulin resistance after vitamin D replacement. The objective of the current study was to examine whether vitamin D replacement improves insulin sensitivity., Methods: In this randomized double-blind placebo-controlled study, 12 healthy subjects with plasma 25-hydroxy vitamin D (25[OH]D) levels of less than 20 ng/mL were treated with ergocalciferol, 50,000 units, orally once a week for 8 weeks or matching placebo. The main outcome measure was insulin-stimulated glucose infusion rate during the last 20 minutes of a hyperinsulinemic-euglycemic glucose clamp study., Results: At baseline, plasma 25(OH)D levels were similar between the ergocalciferol and placebo groups (13.3 ± 3.8 and 15.7 ± 2.4 ng/mL, respectively; P = 0.3) but were higher in the ergocalciferol group at 8 weeks (18.8 ± 5 vs 12.5 ± 2.2 ng/mL; P = 0.02). Glucose infusion rate was similar between the 2 groups both at baseline and after 8 weeks of treatment. Changes in plasma 25(OH)D levels did not correlate with change in glucose infusion rate., Conclusion: Administration of ergocalciferol, 50,000 units, weekly for 8 weeks in subjects with low vitamin D levels improves 25(OH)D levels but does not improve insulin sensitivity.

Objectives: To evaluate the newly developed Roche MODULAR Analytics E170 Total Vitamin D and the Siemens ADVIA Centaur Vitamin D Total assays., Materials and Methods: Assays were evaluated using the Clinical and Laboratory Standards Institute protocols. Split patient samples were compared with LC-MS/MS and DiaSorin LIAISON assays (n=79 including 15 specimens with detectable endogenous 25-OH vitamin D(2)). Assay accuracy was also evaluated using the Vitamin D External Quality Assessment Scheme (DEQAS) samples., Results: The ADVIA Centaur and E170 assays demonstrated maximum total CVs of 14.1% and 5.9%, respectively. Both showed excellent linearity (R(2)>0.99). The ADVIA Centaur assay demonstrated interference with bilirubin at 800 μmol/L, hemolysis at 1.25 g/L, and triglycerides at 2.8 mmol/L. Compared to LC-MS/MS, the ADVIA Centaur assay demonstrated a R(2) value of 0.893, average bias of -8.8%; the E170 assay an R(2) value of 0.872, average bias of 14.3% with underestimation of 25-OH vitamin D(2). Compared to the LIAISON assay, the ADVIA Centaur assay demonstrated an R(2) value of 0.781, average bias of -17.3%; the E170 assay an R(2) value of 0.823, average bias of 11.4%. The ADVIA Centaur and E170 assays demonstrated a biases of <20% in 10/10 and 8/10 DEQAS samples, respectively., Conclusions: The ADVIA Centaur and E170 vitamin D assays demonstrated acceptable linearity, imprecision, and accuracy. The E170 assay demonstrated consistent underestimation of 25-OH vitamin D(2) levels. Compared with LC-MS/MS, the ADVIA Centaur assay demonstrated a higher R(2) value and a smaller average bias than the E170 assay., (Copyright © 2012 The Canadian Society of Clinical Chemists. All rights reserved.)

Few foods contain ergocalciferol or cholecalciferol. Treatment of mushrooms with UV light increases ergocalciferol content and could provide a dietary source of vitamin D. We evaluated the impact of consuming UV-treated white button mushrooms (Agaricus bisporus) on the vitamin D status of healthy adults. Thirty-eight volunteers were randomized to 4 treatments consumed with a standard meal for 6 wk: the control (C) group received untreated mushrooms providing 0.85 μg/d ergocalciferol (n = 10); groups M1 and M2 received UV-treated mushrooms providing 8.8 (n = 10) and 17.1 μg/d (n = 9), respectively; and the supplement (S) group received purified ergocalciferol plus untreated mushrooms, providing a total of 28.2 μg/d (n = 9). Serum total 25-hydroxyvitamin D [25(OH)D] and 25-hydroxyergocalciferol [25(OH)D2] were 83 ± 38 and 2.4 ± 2.0 nmol/L, respectively, at baseline (mean ± SD). At wk 6, 25(OH)D2 had increased and was higher in all treatment groups than in the C group, whereas 25-hydroxycholecalciferol [25(OH)D3] had decreased and was lower in the M2 and S groups than in the C group. Increases in 25(OH)D2 for groups C, M1, M2, and S were 1.2 ± 5.2, 13.8 ± 7.3, 12.7 ± 3.7, and 32.8 ± 3.3 nmol/L and decreases in 25(OH)D3 were -3.9 ± 16.3, -10.4 ± 6.4, -20.6 ± 14.6, and -29.5 ± 15.9 nmol/L, respectively. Concentrations did not change in group C. In summary, ergocalciferol was absorbed and metabolized to 25(OH)D2 but did not affect vitamin D status, because 25(OH)D3 decreased proportionally.

Uncertainty remains regarding the efficacy of low intakes of ergocalciferol (vitamin D2 or D2) and cholecalciferol (vitamin D3 or D3) provided in food to increase serum 25-hydroxy-vitamin D (25-OH-D) metabolite concentrations when UV-B exposure is low. We recruited 40 healthy men and women into a double-blind, parallel design, randomized controlled trial. Participants received placebo or 1 of 4 experimental treatments (D2 or D3 at 5 or 10 μg/d) supplied as a malted milk drink for 4 wk during a period of minimal UV-B exposure in the UK. The primary outcome was a change in serum 25-OH-D2 and 25-OH-D3 concentrations measured by ultra-performance liquid chromatography tandem MS. The secondary outcomes were changes in concentrations of plasma parathyroid hormone and serum calcium (Ca(2+)). Baseline concentrations (geometric mean ± SD) of 25-OH-D2, 25-OH-D3, and total 25-OH-D were 3 ± 4, 32 ± 22, and 37 ± 22 nmol/L, respectively. Both D2- and D3-fortified drinks resulted in dose-dependent increases (P < 0.001) in their respective 25-OH metabolites that did not significantly differ in size. Increments from baseline compared with the placebo group following 5 and 10 μg/d of D2 were (mean ± SEM) 9.4 ± 2.5 and 17.8 ± 2.4 nmol/L for 25-OH-D2 and following 5 and 10 μg/d of D3 were 15.1 ± 4.7 and 22.9 ± 4.6 nmol/L for 25-OH-D3, respectively. There was no difference between D2 and D3 groups in the incremental AUC of their respective metabolites. These findings suggest that D2 and D3 are equipotent in increasing 25-OH-D in healthy men and women with negligible UV-B exposure.

Background: Factors affecting bone calcium deposition across pregnancy and lactation are not well characterized., Objective: The impact of maternal age, calcium intake, race-ethnicity, and vitamin D status on the rate of bone calcium deposition (VO+) was assessed across pregnancy and lactation., Design: Stable calcium isotopes were given to 46 women at pre- or early pregnancy (trimester 1), late pregnancy (trimester 3), and 3-10 wk postpartum. Three cohorts were included: 23 adolescents from Baltimore (MD), aged 16.5 ± 1.4 y (mean ± SD; Baltimore cohort); 13 adults from California, aged 29.5 ± 2.6 y (California cohort); and 10 adults from Brazil, aged 30.4 ± 4.0 y (Brazil cohort). The total exchangeable calcium pool, VO+, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D [1,25(OH)₂D], parathyroid hormone, and calcium intake were evaluated., Results: At trimester 3, inverse associations between 1,25(OH)₂D and VO+ were evident in the Baltimore (P = 0.059) and Brazil (P = 0.008) cohorts and in the whole group (P = 0.029); calcium intake was not a significant determinant of VO+ in any group during pregnancy. At postpartum, a significant positive association was evident between VO+ and calcium intake (P ≤ 0.002) and between VO+ and African ethnicity (P ≤ 0.004) in the whole group and within the Baltimore and Brazil cohorts., Conclusions: Elevated 1,25(OH)₂D was associated with decreased rates of bone calcium deposition during late pregnancy, a finding that was particularly evident in pregnant adolescents and adult women with low calcium intakes. Higher dietary calcium intakes and African ethnicity were associated with elevated rates of bone calcium deposition in the postpartum period.

Background: Several studies suggest that 24,25-dihydroxyvitamin D [24,25(OH)₂D] may have an effect on bone mass and metabolism., Objective: We evaluated the relationship between serum 24,25(OH)₂D levels and bone density and bone metabolism in children with a primary bone disorder-osteogenesis imperfecta (OI)., Materials and Methods: The study included 132 patients (age, 1.1 to 17.9 yr; 67 girls) with OI types I, III, or IV who had not received bisphosphonate treatment at the time of analysis., Results: Serum 24,25(OH)₂D levels were significantly higher in OI type III than in OI type I or IV. Serum 24,25(OH)₂D concentrations were positively correlated with serum 25-hydroxyvitamin D (25OHD) levels and negatively correlated with serum PTH levels, and were not correlated with serum 1α,25-dihydroxyvitamin D [1,25(OH)₂D]. The ratio between serum 24,25(OH)₂D and 25OHD was negatively correlated with age and was independent of serum 25OHD concentrations. Regression analysis revealed that OI severity (P = 0.04), serum 25OHD levels (P < 0.001), and serum PTH concentrations (P = 0.045), but not age, gender, or serum 1,25(OH)₂D, were independent predictors of serum 24,25(OH)₂D levels. No correlation was found between serum 24,25(OH)₂D levels or the ratio between serum 24,25(OH)₂D and 25OHD and lumbar spine bone mineral density z-scores or bone marker levels (serum osteocalcin and urinary collagen type I N-telopeptide) after adjusting for OI type, age, and gender., Conclusion: Patients with more severe OI type had higher 24,25(OH)₂D serum levels and higher serum 24,25(OH)₂D to 25OHD ratios, suggesting an increased 25OHD-24-hydroxylase activity.

Background: Increased risk of fractures and osteoporosis have been associated with the use of antiretroviral drugs. There is a paucity of prospective evaluations of bone markers after the initiation of drugs currently recommended to treat HIV infection and results on the evolution of these markers are conflicting. Lastly, the effect of tenofovir on 1,25-(OH)₂ vitamin D is uncertain., Methods: We performed a prospective study on the evolution of bone markers, parathormone and 1,25-(OH)₂ vitamin D before and after standard antiretroviral regimens. This was a sub-study of a trial conducted in antiretroviral-naïve patients randomized to tenofovir + emtricitabine in combination with either atazanavir/ritonavir (ATV/r) or efavirenz (EFV). Follow-up lasted 48 weeks. The following bone markers were analyzed: C-terminal cross-laps (CTx), osteocalcin (OC), osteoprotegerin (OPG), and receptor activator of nuclear factor κB ligand (RANKL). Mixed-factorial analysis of variance with random-coefficient general linear model was used to compare their trends over time and linear multivariable regression was performed with a backward selection method to assess predictors of their variations from baseline to week 48. Trends of parathormone and 1,25-(OH)₂ vitamin D were also evaluated., Results: Seventy-five patients were studied: 33 received EFV and 42 ATV/r. Significant increases were found for all markers except for RANKL. There was a significant direct association between CTx and OC increases. Multivariable analysis showed that higher glomerular filtration rate (estimated through cystatin C clearance) predicted greater OPG increase, while older age, higher HIV RNA at baseline and use of ATV/r predicted greater CTx increase. A significant increase of parathormone accompanied the evolution of the study markers. 1,25-(OH)₂ vitamin D remained stable, though a seasonality variation was demonstrated., Conclusions: These data demonstrate CTx increase (bone resorption marker) corresponding to OC increase (bone formation marker) early upon HAART initiation. Moreover, predictors of bone marker increases have been suggested, possibly indicating that a stricter monitoring of bone health and pro-active interventions are needed in older patients, those with higher HIV RNA, prescribed ATV/r rather than EFV, and with decreased renal function at baseline. Further studies are needed to clarify the mechanisms responsible for up-regulation of bone turnover markers, as well as to understand if and what markers are best correlated or predictive of pathological fractures.

Background: 1α,25-dihydroxy vitamin D [1,25(OH)(2)D] is the active metabolite of vitamin D. Antibody-based detection methods lack specificity, but when combined with isotope dilution/ultra-performance liquid chromatography (UPLC)-tandem mass spectrometry, immunoextraction provides an attractive method for 1,25(OH)(2)D. We developed a method for simultaneous quantification of 1,25(OH)(2)D(2) and 1,25(OH)(2)D(3) with a 4.6-min instrument cycle time. Results are available 36 h after sample preparation begins., Methods: Sample preparation consisted of protein precipitation, immunoextraction with solid-phase anti-1,25(OH)(2)D antibody, and derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione. Analytes were resolved using reversed-phase UPLC and quantified using positive ion electrospray ionization-tandem mass spectrometry. We used hexadeuterated 1,25(OH)(2)D(3) and 1,25(OH)(2)D(2) as internal standards and performed method comparisons against the DiaSorin RIA and an LC-MS/MS method available at a reference laboratory., Results: 1,25(OH)(2)D(3) intraassay and interassay imprecision was 5.6% and 8.0% (120 pmol/L) and 8.7% and 13% (48 pmol/L). Limits of detection and quantification were 1.5 pmol/L and 3.0 pmol/L, respectively. 1,25(OH)(2)D(2) intraassay and interassay imprecision was 8.7% and 11% (186 pmol/L) and 11% and 13% (58 pmol/L). Limits of detection and quantification were both 1.5 pmol/L. Comparison with RIA had a proportional bias of 0.75, constant bias of -4.1, and Pearson correlation (r(2)) of 0.31. Comparison with a reference LC-MS/MS assay had a proportional bias of 0.89, constant bias of 3.7, and r(2) of 0.88., Conclusions: Protein precipitation with antibody-based extraction is effective for sample preparation before LC-MS/MS analysis of derivatized 1,25(OH)(2)D. This method appears to have improved specificity over a clinically used RIA with low imprecision and limits of detection.

Context: Young women with anorexia nervosa (AN) have a normal vitamin D status. The bioavailability of vitamin D during malnutrition is unknown., Objective: The objective of the study was to examine the serum response to oral ergocalciferol in AN., Design/setting: This was a prospective cohort study, conducted in 2007-2009 at a tertiary care center., Patients/interventions: Twelve adolescents with AN (age 19.6 ± 2.0 yr, body mass index 16.5 ± 1.4 kg/m²) and 12 matched healthy controls (20.0 ± 2.4 yr, 22.7 ± 1.0 kg/m²) received one baseline 50,000 IU oral dose of ergocalciferol., Main Outcomes: Serum D₂, D₃, 25-hydroxyvitamin D, and 1,25-dihydroxyvitamin D, collected before ingestion, at 6 and 24 h and weekly for 4 wk, and body composition measures were measured., Results: The AN group was severely malnourished (77.2 ± 6.3% median body weight), whereas the control group was normal weighted (106.2 ± 6.2%). From a common baseline D₂ (1.5 ± 1.6 nmol/liter, P =0.34) the groups diverged (time × group interaction P = 0.04), peaking at 70 ± 34 nmol/liter at 6 h in controls compared with 43 ± 28 nmol/liter in AN subjects (P = 0.008). The D₂ trajectories converged at 24 h (57 nmol/liter, P = 0.98) and returned to near baseline at 1 wk. Baseline D₃ was higher in AN subjects (12.1 ± 9.6 vs. 3.1 ± 2.3 nmol/liter, P < 0.001) and remained higher throughout. 25-Hydroxyvitamin D followed a common trajectory (time × group interaction P = 0.15), rising to 45 ± 10 nmol/liter at 24 h but returning to baseline by wk 3 (P = 0.36). Correlating vitamin D levels with fat measures (body mass index, body fat) produced similar findings., Conclusions: Despite severe malnutrition, young women with AN had a similar bioavailability of oral ergocalciferol as the healthy-weighted controls. Vitamin D dosing for patients suffering from malnutrition may not differ from that for normal-weighted adolescents.

We have developed an automated high-throughput assay for the determination of vitamin A (retinol), ergocalciferol (25-OH D2), cholecalciferol (25-OH D3) and vitamin E (α-tocopherol) in a small volume of human plasma. Sample preparation involved mixing 50 μL of plasma with 100 μL of ethanol containing isotope-labelled internal standards, followed by mixing with isooctane/chloroform (3:1, 300 μL). The organic phase was evaporated, and the sample reconstituted in 50 μL methanol. The analysis was performed using reversed-phase liquid chromatography with a gradient mobile phase containing water, methanol and ammonium formate. Chromatographic run-time was 5 min, and positive mode electrospray tandem mass spectrometry (MS/MS) was used for detection. The limits of detection were 0.10 μM for all-trans retinol and 3.3 nM for 25-OH D2 and 25-OH D3. Recoveries were 91.9-105.0%, and within- and between-day coefficients of variance (CVs) 2.4-5.3 and 3.1-8.2, respectively. The assay is presently being used in large-scale studies., (Copyright © 2011 John Wiley & Sons, Ltd.)

Vitamin D exists in 2 forms that are important regarding vitamin D status and supply in cattle: vitamin D(2) (D(2)) and vitamin D(3) (D(3)). To become physiologically active, both D(2) and D(3) must undergo 25-hydroxylation in the liver. The resulting 25-hydroxyvitamin D(2) [25(OH)D(2)] and 25-hydroxyvitamin D(3) [25(OH)D(3)] are measured as indicators of the physiological vitamin D status of cattle. The study used 14 Danish Holstein cows housed without access to sunlight. The cows were orally administered 250 mg (1.0 × 10(7)IU) of D(2) and D(3) in a cross-over design with 2 treatment groups and 2 study periods, rendering 4 treatments when carryover effects were taken into account: D(2) given first, D(2) given last after D(3), D(3) given first, and D(3) given last after D(2). Two weeks elapsed between the treatment in the first study period and the treatment in the second study period. Blood samples were collected 0, 3, 6, 14, 17, 20, 23, 26, 40, 48, 70, 94, 166, and 214 h after providing the oral bolus of vitamin to the cows. Comparisons between plasma levels of the metabolites D(2), D(3), 25(OH)D(2), and 25(OH)D(3) over time were made by comparing areas under the plasma concentration curves. Oral administration of D(3) increased plasma D(3) (182.6±17.1 ng/mL; mean ± SEM) and 25(OH)D(3) (103.5±10.0 ng/mL) more efficiently than oral administration of D(2) increased plasma D(2) (49.1±32.6 ng/mL) and 25(OH)D(2) (27.9±2.1 ng/mL). The D(3) given after an oral dose of D(2) was less efficient for increasing plasma concentrations of 25(OH)D(3) (61.2±12.0 ng/mL) compared with D(3) given without previous D(2) administration (103.5±10.0 ng/mL), whereas the plasma concentrations of D(3) itself were the same when given first (182.6±17.1 ng/mL) as when given after D(2) (200.0±123.9 ng/mL). The same occurred for plasma concentrations of D(2) metabolites both if D(2) was given first (49.1±32.6 ng/mL) and after D(3) (54.7±7.7 ng/mL). In conclusion, D(3) given after D(2) is less efficient at increasing the plasma status of 25(OH)D(3) than D(3) given without previous D(2) administration., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Vitamin D therapy is widely used for the treatment of hyperparathyroidism associated with chronic renal failure in renal disease patients. The vitamin D prodrug, 1α-hydroxyvitamin D(2) (1α(OH)D(2)), is used for the treatment of the end stage renal disease patients who as a result of impaired kidney function cannot convert the naturally occurring vitamin D to the active hormonal form namely 1,25-dihydroxyvitamin D(2) (1,25(OH)(2)D(2)). The systemic circulating levels of this active form are in the pg/mL range and represent a significant bioanalytical challenge for therapeutic monitoring. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) is considered the gold standard for the selective and sensitive determination of small molecule therapeutics in biological matrices. However, the reported LC-MS/MS bioanalytical assays for 1,25(OH)(2)D(2) suffer from extensive sample preparation procedures or derivatization protocols to achieve the requisite sensitivity and selectivity. In this paper, we describe an assay that employs 96-well plate solid phase extraction sample preparation combined with highly sensitive LC-MS/MS instrumentation. The utility of ultra high pressure liquid chromatography to reduce the analytical run time was also demonstrated. Employing this assay a lower limit of quantitation of 25.0 pg/mL using 300 μL sample aliquot of rat serum was achieved with linearity obtained over the range of 25.0-1000 pg/mL. Both intra-day and inter-day coefficients of variation were <15% and accuracy across the assay range was within 100±7.24%. The application of the assay was demonstrated for the analysis of 1,25(OH)(2)D(2) rat serum samples to support pharmacokinetic studies conducted at doses down to sub-microgram per kilogram of 1α(OH)D(2)., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background/objectives: The hallmark of cystic fibrosis (CF) is chronic lung inflammation. The severity of lung disease is closely correlated with immunoglobulin G (IgG) levels. Beyond its contribution to the bone health, the importance of vitamin D has not been fully recognized owing to the lack of human studies providing evidence of its benefit. In the context of the recently described immunomodulatory functions of vitamin D, we aimed to assess the relationship between vitamin D and IgG levels., Subjects/methods: Eight hundred and ninety-six CF patients were included (0.53-65.9 years) from seven centers in Denmark, Norway and Sweden. Serum 25-hydroxyvitamin D (25OHD) and total IgG were measured, spirometry was carried out and vitamin D intake data were gathered using a 7-day dietary food record. Multiple linear regression analyses were performed for IgG and forced expiratory volume in 1λs (FEV1) as dependent variables, and serum 25OHD, daily food and supplemented vitamin D sources of intake as independent variables. The model was controlled for age, gender, genotype, CF-related diabetes, season, infection/colonization status, long-term oral corticosteroid treatment, long-term treatment with macrolide antibiotics, pancreatic insufficient phenotype and body mass index z-score., Results: Serum total IgG levels were negatively associated with serum 25OHD (adjusted R (2) = 0.376; beta = -0.02; P<0.001), supplemented vitamin D intake per kg bodyweight (adjusted R (2) = 0.375; beta = -0.82; P < 0.001) and total vitamin D intake per kg bodyweight (adjusted R (2) = 0.398; beta = -0.60; P = 0.002). Serum 25OHD was positively associated with FEV1 (adjusted R (2) = 0.308; beta = 0.0007; P = 0.025)., Conclusions: Increasing vitamin D intake may positively modulate inflammation in CF. This study supports the proposed role of vitamin D in the immune system during infection and substantiates prospective studies.

Context: Vitamin D deficiency and frailty are common with aging, but the association between these conditions is uncertain., Objective: To determine the association between 25-hydroxyvitamin D (25(OH)D) levels and prevalent and incident frailty status among older women., Design: Cross-sectional and longitudinal analyses of a prospective cohort study., Setting: Four U.S. centers., Participants: 6307 women aged≥69 years., Main Outcome Measures: Frailty status classified as robust, intermediate stage, or frail at baseline; and robust, intermediate stage, frail, or dead (all-cause mortality) at follow-up an average of 4.5 years later., Results: At baseline, there was a U-shaped association between 25(OH)D level and odds of frailty with the lowest risk among women with levels 20.0-29.9 ng/ml (referent group). Compared with this group, the odds of frailty were higher among those with levels<15.0 ng/ml [multivariable odds ratio (MOR) 1.47, 95% confidence interval (CI), 1.19-1.82], those with levels 15.0-19.9 ng/ml (MOR 1.24, 95% CI 0.99-1.54), and those with levels≥30 ng/ml (MOR 1.32, 95% CI 1.06-1.63). Among 4551 nonfrail women at baseline, the odds of frailty/death (vs. robust/intermediate) at follow-up appeared higher among those with levels 15.0-19.9 ng/ml (MOR 1.21, 95% CI 0.99-1.49), but the CI overlapped 1.0. The odds of death (vs. robust/intermediate/frail at follow-up) was higher among those with levels<15.0 ng/ml (MOR 1.40, 95% CI 1.04-1.88) and those with levels 15.0-19.9 ng/ml (MOR 1.30, 95% CI 0.97-1.75), although the latter association did not quite reach significance., Conclusion: Lower (<20 ng/ml) and higher (≥30 ng/ml) levels of 25(OH)D among older women were moderately associated with a higher odds of frailty at baseline. Among nonfrail women at baseline, lower levels (<20 ng/ml) were modestly associated with an increased risk of incident frailty or death at follow-up.

The ruminal fate of the fat-soluble vitamins D and E was studied in dairy cows. Ten to 15 kg of ruminal contents was taken from each cow through a ruminal fistula. A sample was taken out (0-h sample) and the rest of the contents were mixed with 4,360 mg of all-rac-α-tocopheryl acetate (vitamin E; study 1) or 4,360 mg of all-rac-α-tocopheryl acetate, 250 mg of ergocalciferol (vitamin D(2)), and 250 mg of cholecalciferol (vitamin D(3); study 2). After mixing, the ruminal contents were returned to the respective cows. Blood was collected 0, 6, 24, and 30 h after introducing the vitamins into the rumen. Samples of ruminal contents were collected at 0, 1, 2, 4, 6, 24, and 30 h (in vivo). From the 1-h sample, 6 subsamples from each cow were incubated at 37 °C and taken out at 2, 4, 6, 12, 24, and 30 h (in vitro). In vivo concentrations of added α-tocopherol, ergocalciferol, and cholecalciferol in the rumen first increased and subsequently declined due to dilution effects of eating and passage out of the rumen. The level of the free-alcohol form of α-tocopherol from the natural content in feed was constant throughout the in vivo study, in contrast to the content of total α-tocopherol, which indicated that no hydrolysis of the acetate form into alcohol form happened in the rumen. In vitro, all added vitamins were found at constant levels; hence, none of the added vitamins were degraded in ruminal contents. The concentration of α-tocopherol in plasma increased at a rate per milligram of ruminally introduced α-tocopherol below the rate of the increase in plasma ergocalciferol or cholecalciferol metabolites per milligram of introduced ergocalciferol or cholecalciferol, respectively, over 24h. In conclusion, ergocalciferol, cholecalciferol, and α-tocopheryl acetate proved to be stable in the rumen and in ruminal contents from high-yielding dairy cows. Changes in plasma concentrations of the vitamins relative to the amount of vitamin introduced to the rumen indicated a lower effect on plasma status of ergocalciferol than of cholecalciferol, and an even lower effect of α-tocopherol. The limited plasma response after a single dose of α-tocopheryl acetate led to the conclusion that oral single dose therapy with all-rac-α-tocopheryl acetate is of limited physiological value., (Copyright © 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Background: Vitamin D has been added to calcium-fortified orange juice. It is unknown whether vitamin D is as bioavailable from orange juice as it is from supplements., Objectives: The objective was to compare the bioavailability of vitamin D(2) and vitamin D(3) from orange juice with that from vitamin D(2) and vitamin D(3) supplements. A secondary aim was to determine which form of vitamin D is more bioavailable in orange juice., Design: A randomized, placebo-controlled, double-blind study was conducted in healthy adults aged 18-84 y (15-20/group) who received 1000 IU vitamin D(3), 1000 IU vitamin D(2), or placebo in orange juice or capsule for 11 wk at the end of winter., Results: A total of 64% of subjects began the study deficient in vitamin D (ie, 25-hydroxyvitamin D [25(OH)D]) concentrations <20 ng/mL). Analysis of the area under the curve showed no significant difference in serum 25(OH)D between subjects who consumed vitamin D-fortified orange juice and those who consumed vitamin D supplements (P = 0.084). No significant difference in serum 25(OH)D(3) was observed between subjects who consumed vitamin D(3)-fortified orange juice and vitamin D(3) capsules (P > 0.1). Similarly, no significant difference in serum 25(OH)D(2) was observed between subjects who consumed vitamin D(2)-fortified orange juice and vitamin D(2) capsules (P > 0.1). No significant overall difference in parathyroid hormone concentrations was observed between the groups (P = 0.82)., Conclusion: Vitamin D(2) and vitamin D(3) are equally bioavailable in orange juice and capsules.

Evidence suggests low vitamin D and elevated parathyroid hormone (PTH) concentrations may increase risk for cardiovascular disease. However, little is known about the association between vitamin D or PTH and subclinical atherosclerosis. This cross-sectional study included 654 community-dwelling older adults aged 55-96 years (mean age, 75.5 years) without a history of coronary heart disease, revascularization, or stroke enrolled in the Rancho Bernardo Study who completed a clinic examination in 1997-1999 and provided a blood sample for determination of serum 25-hydroxyvitamin D [25(OH)D], 1,25-dihydroxyvitamin D [1,25(OH)(2)D], and PTH concentrations. Carotid artery intima-media wall thickness (IMT) was measured as an indicator of atherosclerosis at two sites with B-mode ultrasound. After adjusting for age, sex, smoking, alcohol intake, waist-to-hip ratio, exercise, season of blood draw, diabetes, and hypertension, geometric mean internal carotid IMT (p(trend) 0.022), but not common carotid IMT (p(trend) 0.834) decreased in a dose-dependent fashion with increasing concentration of 25(OH)D. There was no association of 1,25(OH)(2)D or PTH with either measure of carotid IMT. In subgroup analyses, 1,25(OH)(2)D was inversely associated with internal carotid IMT among those with hypertension (p for interaction 0.036). These findings from a population-based cohort of older adults suggest a potential role for vitamin D in the development of subclinical atherosclerosis. Additional research is needed to determine whether vitamin D may influence the progression of atherosclerosis, including the effects of supplementation on the atherosclerotic process.

1Alpha,25(OH)(2) vitamin D(3) [1,25(OH)(2)D(3)] increases serum Ca(2+) concentration in vivo, an action counteracted by activation of the Ca(2+)-sensing receptor (CaSR), which decreases parathyroid hormone (PTH) secretion and increases renal Ca(2+) excretion. Relatively little is known of the role the CaSR plays in this response through its potentially direct actions in kidney, gut, and bone independently of PTH. We report PTH-independent roles of the CaSR in modulating the response to exogenous 1,25(OH)(2)D(3) in mice with targeted disruption of both the CaSR and PTH genes (C(-)P(-)) compared with that in mice with disruption of the PTH gene alone (C(+)P(-)) or wild-type mice (C(+)P(+)). After intraperitoneal injection of 0.5 ng/g body wt 1,25(OH)(2)D(3), peak calcemic responses were observed at 24 h in all three genotypes in association with 1) a greater increase in serum Ca(2+) in C(-)P(-) mice than in the other genotypes on a Ca(2+)-replete diet that was attenuated by a Ca(2+)-deficient diet and pamidronate, 2) increased urinary Ca(2+)-to-creatinine ratios (UCa/Cr) in the C(+)P(-) and C(+)P(+) mice but a lowered ratio in the C(-)P(-) mice on a Ca(2+)-replete diet, and 3) no increase in calcitonin (CT) secretion in the C(+)P(+) and C(+)P(-) mice and a small increase in the C(-)P(-) mice. PTH deficiency had the anticipated effects on the expression of key genes involved in Ca(2+) transport at baseline in the duodenum and kidney, and injection of 1,25(OH)(2)D(3) increased gene expression 8 h later. However, the changes in the genes evaluated did not fully explain the differences in serum Ca(2+) seen among the genotypes. In conclusion, mice lacking the full-length CaSR have increased sensitivity to the calcemic action of 1,25(OH)(2)D(3) in the setting of PTH deficiency. This is principally from enhanced 1,25(OH)(2)D(3)-mediated gut Ca(2+) absorption and decreased renal Ca(2+) excretion, without any differences in bone-related release of Ca(2+) or CT secretion among the three genotypes that could explain the differences in their calcemic responses.

Approximately 10-80% of patients with Cystic Fibrosis (CF) have vitamin D deficiency. Obtaining therapeutic vitamin D levels has been a challenge for CF care providers using current recommended high-dose oral ergocalciferol (400,000 IU over 2 months). The objective of this study was to evaluate the safety and efficacy of a 2-week, very high dose ergocalciferol (700,000 IU over 14 days) repletion strategy in children and young adults with CF. As part of a quality improvement initiative, a prospective cohort study was performed from January through May 2007. Phase I included identifying individuals with CF who were subtherapeutic in 25-OH D. In phase II, 50,000 IU of ergocalciferol was prescribed for a 14 day term and administered daily. During phase III, a post treatment 25-OH D level was obtained to determine improvement. Baseline demographics and clinical characteristics were obtained at study entry. Stratification of the post 25-OHD levels was defined. Eighteen individuals with CF participated in the study. The mean age was 17+/-5 years (range 6-25 years). One hundred percent were pancreatic insufficient and required pancreatic enzyme replacement. All 18 had 25-OHD levels less than 30 ng/mL pre-treatment. Seventeen of the 18 (94%) participants became therapeutic in the 2-week interval. No patients had values considered high abnormal (100-150 ng/mL) or toxic (>150 ng/mL). Mean change was noted at an increase of 37.3+/-22 ng/mL in the 2-week period (p<0.001). Pre and peripubertal individuals had a significantly greater increase in 25-OH D levels. The results of this study demonstrate that very high dosing of vitamin D using oral ergocalciferol over a 14 day period is an effective strategy in achieving therapeutic levels of 25-OH vitamin D in children and young adults with CF. We believe this regimen deserves further study.

Background: The effects of vitamin D2 and D3 supplementation on circulating concentrations of 25(OH)D3 require reliable analytical tools for specific determination of 25(OH)D3 and 25(OH)D2. We have developed a highly specific 25-OH Vitamin D3 ELISA with negligible cross-reactivity towards 25(OH)D2., Methods: 25(OH)D3 concentrations were measured in several study participants; 1) 641 healthy men and women; 2) 39 postmenopausal women receiving 400-800 IU vitamin D3 daily for 4 months; 3) 45 men and women with hip fracture receiving 1000 IU vitamin D2 daily for 3 months., Results: This 25-OH Vitamin D3 ELISA had minimal cross-reactivity to 25(OH)D2, (0.7%), and demonstrated a high correlation (r2 = 0.93) with 25(OH)D3 determined by HPLC. 25(OH)D3 increased by 14% in subjects receiving vitamin D3 for 4 months (p < 0.01), whereas there was no significant change in 25(OH)D3 levels in those receiving vitamin D2., Conclusions: We report that 25(OH)D3 ELISA was used for evaluation of 25(OH)D3 concentrations in subjects receiving vitamin D2 and D3 supplementation. The increase of 25(OH)D3 in circulation with vitamin D3 supplementation and lack of increase with vitamin D2 supplementation suggest that this assay has sufficient sensitivity and specificity to be used as a reliable measurement of nutritional vitamin D3 status in humans.

Background: Vitamin D is important for bone health and immune regulation, and has been shown to be low in multiple sclerosis (MS). We sought to determine the effect of over the counter low dose cholecalciferol (LDC) and high dose ergocalciferol (HDE) on the vitamin D levels in MS patients., Methods: We retrospectively evaluated serum 25-hydroxy-vitamin D [25(OH)D] levels of 199 patients (CIS, n = 32; RRMS, n = 115; PPMS, n = 10; SPMS, n = 16; Transverse Myelitis (TM), n = 9; other neurological diseases, n = 16) attending our clinic between 2004 and 2008. We examined the change in 25(OH)D levels in 40 MS patients who took either LDC (< or =800 IU/day) or HDE (50,000 IU/day for 7-10 days, followed by 50,000 IU weekly or biweekly)., Results: The average 25(OH)D level was 71 +/- 39 nmol/L (Mean +/- SD), and 167(84%) patients had insufficient levels (< or =100 nmol/L) of 25(OH)D. The patients supplemented with LDC did not have a significant increase in their 25(OH)D levels. However, 25(OH)D levels increased by 42 nmol/L (P = 0.01) in the patients originally taking LDC and then prescribed HDE. Optimal levels (> or =100 nmol/L) were only achieved in less than 40% of patients., Conclusions: We conclude that large numbers of patients with MS and TM in our cohort are deficient in vitamin D. HDE significantly elevated 25(OH)D levels in MS patients and was more effective at increasing 25(OH)D levels than LDC. Prospective studies are required to determine appropriate dosing regimen to achieve optimal levels in the majority of MS patients and to ascertain the safety, immunological response, and ultimately the clinical efficacy of vitamin D replacement therapy.