Differential Effects of Oral Boluses of Vitamin D2 vs Vitamin D3 on Vitamin D Metabolism: A Randomized Controlled Trial.

CONTEXT: Vitamin D2 and vitamin D3 have been hypothesized to exert differential effects on vitamin D metabolism. OBJECTIVE: To compare the influence of administering vitamin D2 vs vitamin D3 on metabolism of vitamin D3. METHODS: We measured baseline and 4-month serum concentrations of vitamin D3, 25-hydroxyvitamin D3 [25(OH)D3], 25-hydroxyvitamin D2, 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3], 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], and 4ß,25-dihydroxyvitamin D3 [4ß,25(OH)2D3] in 52 adults randomized to receive a total of four oral bolus doses of 2.5 mg vitamin D2 (n = 28) or vitamin D3 (n = 24) over four months. Metabolite-to-parent compound ratios were calculated to estimate hydroxylase activity. Pairwise before vs after comparisons were made to evaluate effects of vitamin D2 and vitamin D3 on metabolism of vitamin D. Mean postsupplementation metabolite-to-parent ratios were then compared between groups. RESULTS: Vitamin D2 was less effective than vitamin D3 in elevating total serum 25(OH)D concentration. Vitamin D2 suppressed mean four-month serum concentrations of 25(OH)D3, 24R,25(OH)2D3, 1α,25(OH)2D3, and 4ß,25(OH)2D3 and mean ratios of 25(OH)D3 to D3 and 1α,25(OH)2D3 to 25(OH)D3, while increasing the mean ratio of 24R,25(OH)2D3 to 25(OH)D3. Vitamin D3 increased mean four-month serum concentrations of 25(OH)D3, 24R,25(OH)2D3, 1α,25(OH)2D3, and 4ß,25(OH)2D3 and the mean ratio of 24R,25(OH)2D3 to 25(OH)D3. Participants receiving vitamin D2 had lower mean postsupplementation ratios of 25(OH)D3 to vitamin D3 and 1α,25(OH)2D3 to 25(OH)D3 than those receiving vitamin D3. Mean postsupplementation ratios of 24R,25(OH)2D3 to 25(OH)D3 and 4ß,25(OH)2D3 to 25(OH)D3 did not differ between groups. CONCLUSIONS: Bolus-dose vitamin D2 is less effective than bolus-dose vitamin D3 in elevating total serum 25(OH)D concentration. Administration of vitamin D2 reduces 25-hydroxylation of vitamin D3 and 1-α hydroxylation of 25(OH)D3, while increasing 24R-hydroxylation of 25(OH)D3.

Longitudinal changes in serum vitamin D binding protein and free 25-hydroxyvitamin D in a multiracial cohort of pregnant adolescents.

Serum free 25-hydroxyvitamin D (25(OH)D) rather than total 25(OH)D may better indicate vitamin D status during pregnancy given the pregnancy-associated increase in serum vitamin D binding protein (DBP) concentration. Our aims were to assess changes in DBP and free 25(OH)D across gestation and to determine whether free compared with total 25(OH)D more strongly correlates with markers of vitamin D and calcium metabolism during pregnancy. This ancillary study included 58 pregnant adolescents (53% African American, 47% White) who completed a vitamin D3 supplementation study in Rochester, NY. Blood was collected at entry, mid-study, and delivery (median 17, 29, and 40 weeks' gestation). Mixed-effects regression was used to test for differences in DBP, directly measured free 25(OH)D, and other serum markers by study visit and race. Free and total 25(OH)D were evaluated in relation to serum PTH, 1,25(OH)2D, 24,25(OH)2D, and calcium. The mean DBP concentration was above nonpregnant reference values at entry and increased across gestation (P < 0.0001). Total 25(OH)D explained most of the variance in free 25(OH)D (r ≥ 0.67; P < 0.0001). Holding total 25(OH)D constant, each 100 mg/L increase in DBP was associated with a 0.4 pg/mL decrease in free 25(OH)D (P < 0.01). The percent free 25(OH)D was inversely related to both DBP and total 25(OH)D at each visit. Regardless of race or visit, total 25(OH)D was a stronger correlate of PTH, 1,25(OH)2D, and 24,25(OH)2D, and neither total nor free 25(OH)D was related to serum calcium. African Americans had lower total 25(OH)D (P < 0.0001), but free 25(OH)D did not significantly differ by race (P = 0.2). In pregnant adolescents, DBP concentration was elevated and inversely associated with percent free 25(OH)D, but measured free 25(OH)D provided no advantage over total 25(OH)D as a predictor of PTH, 1,25(OH)2D, 24,25(OH)2D, or calcium. The clinical relevance of the small racial difference in percent free 25(OH)D requires further investigation.

Magnesium status and supplementation influence vitamin D status and metabolism: results from a randomized trial.

Background: Previous in vitro and in vivo studies indicate that enzymes that synthesize and metabolize vitamin D are magnesium dependent. Recent observational studies found that magnesium intake significantly interacted with vitamin D in relation to vitamin D status and risk of mortality. According to NHANES, 79% of US adults do not meet their Recommended Dietary Allowance of magnesium. Objectives: The aim of this study was to test the hypothesis that magnesium supplementation differentially affects vitamin D metabolism dependent on baseline 25-hydroxyvitamin D [25(OH)D] concentration. Methods: The study included 180 participants aged 40-85 y and is a National Cancer Institute independently funded ancillary study, nested within the Personalized Prevention of Colorectal Cancer Trial (PPCCT), which enrolled 250 participants. The PPCCT is a double-blind 2 × 2 factorial randomized controlled trial conducted in the Vanderbilt University Medical Center. Doses for both magnesium and placebo were customized based on baseline dietary intakes. Subjects were randomly assigned to treatments using a permuted-block randomization algorithm. Changes in plasma 25-hydroxyvitamin D3 [25(OH)D3], 25-hydroxyvitamin D2 [25(OH)D2], 1,25-dihydroxyvitamin D3, 1,25-dihydroxyvitamin D2, and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] were measured by liquid chromatography-mass spectrometry. Results: The relations between magnesium treatment and plasma concentrations of 25(OH)D3, 25(OH)D2, and 24,25(OH)2D3 were significantly different dependent on the baseline concentrations of 25(OH)D, and significant interactions persisted after Bonferroni corrections. Magnesium supplementation increased the 25(OH)D3 concentration when baseline 25(OH)D concentrations were close to 30 ng/mL, but decreased it when baseline 25(OH)D was higher (from ∼30 to 50 ng/mL). Magnesium treatment significantly affected 24,25(OH)2D3 concentration when baseline 25(OH)D concentration was 50 ng/mL but not 30 ng/mL. On the other hand, magnesium treatment increased 25(OH)D2 as baseline 25(OH)D increased. Conclusion: Our findings suggest that optimal magnesium status may be important for optimizing 25(OH)D status. This trial was registered at clinicaltrials.gov as NCT03265483.

Comparison of the effect of daily versus bolus dose maternal vitamin D3 supplementation on the 24,25-dihydroxyvitamin D3 to 25-hydroxyvitamin D3 ratio.

OBJECTIVE: Supplementing lactating mothers with high doses of vitamin D3 can adequately meet vitamin D requirements of the breastfed infant. We compared the effect of bolus versus daily vitamin D3 dosing in lactating mothers on vitamin D3 catabolism. We hypothesized that catabolism of 25(OH)D3 to 24,25(OH)2D3 would be greater in the bolus than in the daily dose group. DESIGN, SETTING AND PATIENTS: Randomized controlled trial (clinicaltrials.govNCT01240265) in 40 lactating women. INTERVENTIONS: Subjects were randomized to receive vitamin D3 orally, either a single dose of 150,000IU or 5000IU daily for 28days. Vitamin D metabolites were measured in serum and breast milk at baseline, 1, 3, 7, 14 and 28days. MAIN OUTCOME MEASURE: Temporal changes in the serum 24,25(OH)2D3/25(OH)D3 ratio. RESULTS: The concentration of serum 24,25(OH)2D3 was directly related to that of 25(OH)D in both groups (r2=0.63; p<0.001). The mean (±SD) 24,25(OH)2D3/25(OH)D3 ratio remained lower at all time points than baseline values in the daily dose group (0.093±0.024, 0.084±0.025, 0.083±0.024, 0.080±0.020, 0.081±0.023, 0.083±0.018 at baseline, 1, 3, 7, 14, and 28days, respectively). In the single dose group, the increase in 24,25(OH)2D3 lagged behind that of 25(OH)D, but the 24,25(OH)2D3/25(OH)D3 values (0.098±0.032, 0.067±0.019, 0.081±0.017, 0.092±0.024, 0.103±0.020, 0.106±0.024, respectively) exceeded baseline values at 14 and 28days and were greater than the daily dose group at 14 and 28days (p=0.003). The 24,25(OH)2D3/25(OH)D3 ratio remained in the normal range with both dosing regimens. Greater breast milk vitamin D3 values in the single dose group were inversely associated with the 24,25(OH)2D3/25(OH)D3 ratio (r2=0.14, p<0.001), but not with daily dosing. CONCLUSIONS: After a 14-day lag, a single high dose of vitamin D led to greater production of 24,25(OH)2D3, presumably via induction of the 24-hydroxylase enzyme (CYP24A1), relative to the 25(OH)D3 value than did daily vitamin D supplementation, and this effect persisted for at least 28days after vitamin D administration. A daily dose of vitamin D may have more lasting effectiveness in increasing 25(OH)D3 with lesser diversion of 25(OH)D3 to 24,25(OH)2D3 than does larger bolus dosing.

Dose-response effects of supplementation with calcifediol on serum 25-hydroxyvitamin D status and its metabolites: A randomized controlled trial in older adults.

BACKGROUND & AIMS: Oral supplementation with vitamin D is recommended for older adults to maintain a sufficient 25-hydroxyvitamin D (25(OH)D) status throughout the year. While supplementation with vitamin D2 or D3 is most common, alternative treatment regimens exist which require further investigation with respect to increasing 25(OH)D concentration. We investigated the dose-response effects of supplementation with calcifediol compared to vitamin D3 and assessed the dose which results in mean serum 25(OH)D3 concentrations between 75 and 100 nmol/L. METHODS: This randomized, double-blind intervention study included men and women aged ≥65 years (n = 59). Participants received either 5, 10 or 15 µg calcifediol or 20 µg vitamin D3 per day, for a period of 24 weeks. Blood samples were collected every four weeks to assess response profiles of vitamin D related metabolites; serum vitamin D3, 25(OH)D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3). Further, serum calcium, plasma parathyroid hormone, and urinary calcium were evaluated. RESULTS: Supplementation with 20 µg vitamin D3 increased 25(OH)D3 concentrations towards 70 nmol/L within 16 weeks. Supplementation with 10 or 15 µg calcifediol increased 25(OH)D3 levels >75 nmol/L in 8 and 4 weeks, respectively. Steady state was achieved from week 12 onwards with serum 25(OH)D3 levels stabilizing between 84 and 89 nmol/L in the 10 µg calcifediol group. A significant association was observed between the changes in 25(OH)D3 and 24,25(OH)2D3 (R2 = 0.83, P < 0.01), but not between 25(OH)D3 and 1,25(OH)2D3 (R2 = 0.04, P = 0.18). No cases of hypercalcemia occurred in any treatment during the study period. CONCLUSIONS: Calcifediol supplementation rapidly and safely elevates serum 25(OH)D3 concentrations to improve vitamin D status in older adults. A daily dose of 10 µg calcifediol allows serum 25(OH)D3 concentrations to be maintained between 75 and 100 nmol/L. TRIAL REGISTRATION NUMBER: NCT01868945.

Serum 24,25-dihydroxyvitamin D3 response to native vitamin D2 and D3 Supplementation in patients with chronic kidney disease on hemodialysis.

BACKGROUND & AIMS: While vitamin D deficiency is common in patients with end stage renal disease on dialysis and treatment with Vitamin D2 and Vitamin D3 is becoming increasingly common in these patients, little is known about 24,25(OH)2D3 metabolite production. Some authors report that the CYP24A1 enzyme is upregulated in CKD, but reports of low serum levels of 24,25(OH)2D3 in these patients bring this into question. Lack of substrate or increased clearance of the metabolite have been proposed as possible causes. We report serum 24,25(OH)2D3 levels from three controlled trials of Vitamin D2 and Vitamin D3 supplementation which reached adequate levels of 25(OH)D in patients with end stage renal disease on dialysis. METHODS: 680 samples from three controlled trials of Vitamin D2 or Vitamin D3 supplementation in CKD Stage 5D were available for analysis. The trials used single doses of 50,000 IU Vitamin D3, or 50,000 IU Vitamin D2, or weekly doses of 10,000 IU or 20,000 IU Vitamin D3. Blood samples were drawn at baseline and frequently over the ensuing 3-4 months. Serum 25(OH)D and 24,25(OH)2D3 levels were measured using a novel, very sensitive LC-MS/MS-based method involving derivatization with DMEQ-TAD. Linear mixed effect regression models were used to compare the 3 studies and the interventions within studies over time. RESULTS: The subjects given Vitamin D3 had significant increases in 25(OH)D levels. Serum 24,25(OH)2D3 levels were low at baseline in the renal patients and rose slightly with native vitamin D supplementation, but these levels were lower than reports of 24,25(OH)2D3 in healthy populations. CONCLUSIONS: We conclude that the enzymatic activity of CYP24A1 is abnormal in end stage renal patients on dialysis. These trials were registered on clinicaltrials.govNCT00511225 on 8/1/2007; NCT01325610 on 1/17/2011; and NCT01675557 on 8/28/2012.

Placental vitamin D metabolism and its associations with circulating vitamin D metabolites in pregnant women.

Background: Little is known about placental vitamin D metabolism and its impact on maternal circulating vitamin D concentrations in humans.Objective: This study sought to advance the current understanding of placental vitamin D metabolism and its role in modulating maternal circulating vitamin D metabolites during pregnancy.Design: Nested within a feeding study, 24 healthy pregnant women (26-29 wk of gestation) consumed a single amount of vitamin D (511 IU/d from diet and a cholecalciferol supplement) for 10 wk. Concentrations of placental and blood vitamin D metabolites and placental messenger RNA (mRNA) abundance of vitamin D metabolic pathway components were quantified. In addition, cultured human trophoblasts were incubated with 13C-cholecalciferol to examine the intracellular generation and secretion of vitamin D metabolites along with the regulation of target genes.Results: In placental tissue, 25-hydroxyvitamin D3 [25(OH)D3] was strongly correlated (r = 0.83, P < 0.001) with 24,25-dihydroxyvitamin D3 Moreover, these placental metabolites were strongly correlated (r ≤ 0.85, P ≤ 0.04) with their respective metabolites in maternal circulation. Positive associations (P ≤ 0.045) were also observed between placental mRNA abundance of vitamin D metabolic components and circulating vitamin D metabolites [i.e., LDL-related protein 2 (LRP2, also known as megalin) with 25(OH)D3 and the C3 epimer of 25(OH)D3 [3-epi-25(OH)D3]; cubilin (CUBN) with 25(OH)D3; 25-hydroxylase (CYP2R1) with 3-epi-25(OH)D3; 24-hydroxylase (CYP24A1) with 25(OH)D3, 3-epi-25(OH)D3, and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]; and 1α-hydroxylase [(CYP27B1) with 3-epi-25(OH)D3 and 1,25(OH)2D3]. Notably, in vitro experiments with trophoblasts showed increased production and secretion of 25(OH)D3 and higher CYP24A1 gene transcript abundance in response to cholecalciferol treatment.Conclusions: The numerous associations of many of the placental biomarkers of vitamin D metabolism with circulating vitamin D metabolites among pregnant women [including a CYP27B1-associated increase in 1,25(OH)2D3] and the evidence of trophoblast production and secretion of vitamin D metabolites, especially 25(OH)D3, suggest that the placenta may play an active role in modulating the vitamin D metabolite profile in maternal circulation in human pregnancy. This trial was registered at clinicaltrials.gov as NCT03051867.

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.

Efficacy of High-Dose Vitamin D Supplements for Elite Athletes.

PURPOSE: Supplementation with dietary forms of vitamin D is commonplace in clinical medicine, elite athletic cohorts, and the general population, yet the response of all major vitamin D metabolites to high doses of vitamin D is poorly characterized. We aimed to identify the responses of all major vitamin D metabolites to moderate- and high-dose supplemental vitamin D3. METHODS: A repeated-measures design was implemented in which 46 elite professional European athletes were block randomized based on their basal 25[OH]D concentration into two treatment groups. Athletes received either 35,000 or 70,000 IU·wk vitamin D3 for 12 wk, and 42 athletes completed the trial. Blood samples were collected for 18 wk to monitor the response to supplementation and withdrawal from supplementation. RESULTS: Both doses led to significant increases in serum 25[OH]D, and 1,25[OH]2D3. 70,000 IU·wk also resulted in a significant increase of the metabolite 24,25[OH]2D at weeks 6 and 12 that persisted after supplementation withdrawal at week 18, despite a marked decrease in 1,25[OH]2D3. Intact parathyroid hormone was decreased in both groups by week 6 and remained suppressed throughout the trial. CONCLUSIONS: High-dose vitamin D3 supplementation (70,000 IU·wk) may be detrimental for its intended purposes because of increased 24,25[OH]2D production. Rapid withdrawal from high-dose supplementation may inhibit the bioactivity of 1,25[OH]2D3 as a consequence of sustained increases in 24,25[OH]2D that persist as 25[OH]D and 1,25[OH]2D concentrations decrease. These data imply that lower doses of vitamin D3 ingested frequently may be most appropriate and gradual withdrawal from supplementation as opposed to rapid withdrawal may be favorable.

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.

Chemotyping the distribution of vitamin D metabolites in human serum.

Most studies examining the relationships between vitamin D and disease or health focus on the main 25-hydroxyvitamin D3 (25(OH)D3) metabolite, thus potentially overlooking contributions and dynamic effects of other vitamin D metabolites, the crucial roles of several of which have been previously demonstrated. The ideal assay would determine all relevant high and low-abundant vitamin D species simultaneously. We describe a sensitive quantitative assay for determining the chemotypes of vitamin D metabolites from serum after derivatisation and ultra-high performance liquid chromatography-electrospray ionisation-tandem mass spectrometry (UHPLC-ESI-MS/MS). We performed a validation according to the 'FDA Guidance for Industry Bioanalytical Method Validation'. The proof-of-concept of the method was then demonstrated by following the metabolite concentrations in patients with chronic liver diseases (CLD) during the course of a vitamin D supplementation study. The new quantitative profiling assay provided highly sensitive, precise and accurate chemotypes of the vitamin D metabolic process rather than the usually determined 25(OH)D3 concentrations.

Vitamin D3 supplementation: Response and predictors of vitamin D3 metabolites - A randomized controlled trial.

BACKGROUND & AIMS: Large parts of the population are insufficiently supplied with vitamin D, in particular when endogenous synthesis is absent. Therefore many health care providers recommend the use of vitamin D supplements. The current study aimed to investigate the efficacy of an once-daily oral dose of 20 µg vitamin D3 to improve the vitamin D status and to evaluate predictors of response. METHODS: The study was conducted as a double-blind, randomized, placebo-controlled parallel trial from January till April 2013. In total, 105 subjects (20-71 years) were allocated to receive either a vitamin D3 supplement (20 µg/d) or a placebo for 12 weeks. Circulating levels of vitamin D3 metabolites such as the 25(OH)D3 and the 24,25(OH)2D3, and biomarkers of calcium and phosphate metabolism were quantified. RESULTS: The 25(OH)D3 serum concentrations in the placebo group decreased from 38 ± 15 nmol/L at baseline to 32 ± 14 nmol/L and 32 ± 13 nmol/L at weeks 8 and 12 of the study, respectively (p < 0.01). In the vitamin D3 group, the serum 25(OH)D3 concentration increased from 38 ± 14 nmol/L at baseline to 70 ± 15 nmol/L and 73 ± 16 nmol/L at weeks 8 and 12 of vitamin D3 supplementation (p < 0.001), respectively. As a result, 94% of the vitamin D3-supplemented participants reached 25(OH)D3 concentrations of ≥50 nmol/L and thereof 46% attained 25(OH)D3 levels of ≥75 nmol/L until the end of the study. The extent of the 25(OH)D3 increase upon vitamin D3 supplementation depended on 25(OH)D3 baseline levels, age, body weight and circulating levels of triglycerides. In contrast to 25(OH)D3, the response of 24,25(OH)2D3 to the vitamin D3 treatment was affected only by baseline levels of 24,25(OH)2D3 and age. CONCLUSIONS: The average improvement of 25(OH)D3 levels in individuals who received 20 µg vitamin D3 per day during the winter months was 41 nmol/L compared to individuals without supplementation. As a result almost all participants with the vitamin D3 supplementation attained 25(OH)D3 concentrations of 50 nmol/L and higher. The suitability of 24,25(OH)2D3 as a marker of vitamin D status needs further investigation. Clinical trial registration number at clinicaltrials.gov: NCT01711905.

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.

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.

Modified-release oral calcifediol corrects vitamin D insufficiency with minimal CYP24A1 upregulation.

Vitamin D insufficiency is prevalent in chronic kidney disease (CKD) and associated with secondary hyperparathyroidism (SHPT) and increased risk of bone and vascular disease. Unfortunately, supplementation of stage 3 or 4 CKD patients with currently recommended vitamin D2 or D3 regimens does not reliably restore serum total 25-hydroxyvitamin D to adequacy (≥30ng/mL) or effectively control SHPT. Preclinical and clinical studies were conducted to evaluate whether the effectiveness of vitamin D repletion depends, at least in part, on the rate of repletion. A modified-release (MR) oral formulation of calcifediol (25-hydroxyvitamin D3) was developed which raised serum 25-hydroxyvitamin D3 and calcitriol levels gradually. Single doses of either bolus intravenous (IV) or oral MR calcifediol were administered to vitamin D deficient rats. Bolus IV calcifediol produced rapid increases in serum 25-hydroxyvitamin D3, calcitriol and FGF23, along with significant induction of CYP24A1 in both kidney and parathyroid gland. In contrast, oral MR calcifediol produced gradual increases in serum 25-hydroxyvitamin D3 and calcitriol and achieved similar hormonal exposure, yet neither CYP24A1 nor FGF23 were induced. A 10-fold greater exposure to bolus IV than oral MR calcifediol was required to similarly lower intact parathyroid hormone (iPTH). Single doses of oral MR (450 or 900µg) or bolus IV (450µg) calcifediol were administered to patients with stage 3 or 4 CKD, SHPT and vitamin D insufficiency. Changes in serum 25-hydroxyvitamin D3 and calcitriol and in plasma iPTH were determined at multiple time-points over the following 42 days. IV calcifediol produced abrupt and pronounced increases in serum 25-hydroxyvitamin D3 and calcitriol, but little change in plasma iPTH. As in animals, these surges triggered increased vitamin D catabolism, as evidenced by elevated production of 24,25-dihydroxyvitamin D3. In contrast, MR calcifediol raised serum 25-hydroxyvitamin D3 and calcitriol gradually, and meaningfully lowered plasma iPTH levels. Taken together, these studies indicate that rapid increases in 25-hydroxyvitamin D3 trigger CYP24A1 and FGF23 induction, limiting effective exposure to calcitriol and iPTH reduction in SHPT. They also support further investigation of gradual vitamin D repletion for improved clinical effectiveness. This article is part of a Special Issue entitled "17th Vitamin D Workshop".

Dietary calcium does not interact with vitamin D3 in terms of determining the response and catabolism of serum 25-hydroxyvitamin D during winter in older adults.

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.

Clinical utility of simultaneous quantitation of 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D by LC-MS/MS involving derivatization with DMEQ-TAD.

CONTEXT: The discovery of hypercalcemic diseases due to loss-of-function mutations in 25-hydroxyvitamin D-24-hydroxylase has placed a new demand for sensitive and precise assays for 24,25-dihydroxyvitamin D [24,25-(OH)2D]. OBJECTIVE: We describe a novel liquid chromatography and tandem mass spectrometry-based method involving derivatization with DMEQ-TAD {4-[2-(6,7-dimethoxy-4-methyl-3,4-dihydroquinoxalinyl)ethyl]-1,2,4-triazoline-3,5-dione} to simultaneously assay multiple vitamin D metabolites including 25-hydroxyvitamin D (25-OH-D) and 24,25-(OH)2D using 100 µL of serum with a 5-minute run time. DESIGN: The assay uses a newly synthesized internal standard d6-24,25-(OH)2D3 enabling the quantitation of 24,25-(OH)2D3 as well as the determination of the ratio of 25-OH-D3 to 24,25-(OH)2D3, a physiologically useful parameter. SETTING: We report data on more than 1000 normal and disease samples involving vitamin D deficiency or hypercalcemia in addition to studies involving knockout mouse models. RESULTS: The assay showed good correlation with samples from quality assurance schemes for 25-OH-D (25-OH-D2 and 25-OH-D3) determination (-2% to -5% bias) and exhibited low inter- and intraassay coefficients of variation (4%-7%) and lower limits of quantitation of 0.25-0.45 nmol/L. In clinical studies, we found a strong correlation between serum levels of 25-OH-D3 and 24,25-(OH)2D3 (r(2) = 0.80) in subjects over a broad range of 25-OH-D3 values and a marked lack of production of 24,25-(OH)2D3 below 25 nmol/L of 25-OH-D. The ratio of 25-OH-D3 to 24,25-(OH)2D3, which remained less than 25 in vitamin D-sufficient subjects (serum 25-OH-D < 50 nmol/L) but was greatly elevated (80-100) in patients with idiopathic infantile hypercalcemia. CONCLUSIONS: The new method showed good utility in clinical settings involving vitamin D deficiency; supplementation with vitamin D and idiopathic infantile hypercalcemia, as well as in animal models with ablation of selected cytochrome P450-containing enzymes involved in vitamin D metabolism.

Influence of estrogen therapy on calcium, phosphorus, and other regulatory hormones in postmenopausal women: the MESA study.

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.

1,25(OH)2D3 and specific IgE levels in children with recurrent tonsillitis, and allergic rhinitis.

OBJECTIVES: We investigated 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and specific immunoglobulin E (IgE) levels in children with recurrent tonsillitis (RT) plus allergic rhinitis (AR). METHODS: Thirty children with RT+AR were included in the study group, and 30 healthy children comprised the control group. AR-related symptoms were determined using a symptom scale. 1,25(OH)2D3 and specific IgE measurements were made in both groups. RESULTS: The 1,25(OH)2D3 value was significantly lower in the RT+AR group than in the control group. Specific IgE (mixed) panels were in normal limits in both groups; whereas specific IgE (mixed) grass pollen panel value of RT+AT group was significantly higher than that of the control group. Higher nasal itching, nasal obstruction, and concha edema scores were related to significantly higher specific IgE values for the (mixed) grass pollen panel, whereas higher sneeze scores were related to higher specific IgE values for the (mixed) pediatric panel. CONCLUSIONS: Children with grass pollen allergy may not be exposed to sufficient sunlight. With reduced 1,25(OH)2D3, T helper cells may increase, and allergic response also increases. As allergic events increased, these children did not go outside and thus lacked sun exposure. This vicious cycle must be broken, and children with RT+AR should have sunlight exposure to increase 1,25(OH)2D3 levels.

Long-term bioavailability after a single oral or intramuscular administration of 600,000 IU of ergocalciferol or cholecalciferol: implications for treatment and prophylaxis.

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.