Functionality and 25-hydroxyvitamin D levels in institutionalized older adults / Funcionalidade e níveis de 25-hidroxivitamina D em idosos institucionalizados

OBJECTIVES: To evaluate the frequency of hypovitaminosis D among older adults and its association with the level of functionality. METHODS: This cross-sectional observational study of older adults residing in a non-profit long-term care facility assessed functionality with the Katz Index of Independence in Activities of Daily Living. Vitamin D levels were classified as: deficient (< 20 ng/mL), insufficient (21-29 ng/mL), or normal (≥ 30 ng/mL). We used the chi-square test and Student's t-test to compare dichotomous and continuous variables, respectively. Analysis of variance with Tukey's post hoc test was used to assess differences between groups. RESULTS: The sample consisted of 63 individuals whose mean age was 81 (61-113) years: 36 (55.4%) women and 27 (44.6%) men. The mean vitamin D level was 18.6 ng/mL, being < 30 ng/mL in 84.1%. The level was normal in 10 (15.9%), insufficient in 17 (27%), and deficient in 36 (57.1%). Vitamin D deficiency was present in 76.5% of those with total functional dependence (Katz = 5-6). CONCLUSIONS: We observed a high frequency of hypovitaminosis D, especially vitamin D deficiency, which was very common among those with significant functional dependence.

OBJETIVOS: Avaliar a frequência de hipovitaminose D em idosos de uma instituição filantrópica de longa permanência e sua associação com grau de funcionalidade. METODOLOGIA: Estudo transversal, observacional e analítico de idosos de uma instituição filantrópica de longa permanência. A funcionalidade foi avaliada pela Escala de Katz. Os níveis de vitamina D foram classificados em: deficiência (valores menores que 20 ng/mL); insuficiência (valores entre 21 - 29 ng/mL) e normais (valores igual ou superior a 30 ng/mL). Empregamos teste qui-quadrado e t de student, para compararmos variáveis dicotômicas e contínuas, respectivamente; e análise de variância (ANOVA) com teste post hoc de Tukey, para avaliarmos as diferenças entre os grupos. RESULTADOS: Sessenta e três indivíduos foram analisados com média de idade de 81 anos (61 - 113), sendo 36 (55,4%) mulheres e 27 (44,6%) homens. A média de vitamina D foi 18,6 ng/mL, 84,1% com níveis menores que 30 ng/mL; dez apresentaram níveis normais (15,9%), 17 com insuficiência (27%) e 36 com deficiência (57,1%); ainda, 76,5% dos portadores de dependência funcional total (Katz = 5 - 6) apresentam deficiência de vitamina D. CONCLUSÕES: Observamos uma alta frequência de hipovitaminose D, especialmente deficiência, muito frequentes naqueles com dependência funcional importante

Multiplex LC-MS/MS for simultaneous determination of 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D3, albumin, and vitamin D-binding protein with its isoforms: One-step estimation of bioavailable vitamin D and vitamin D metabolite ratio.

Bioavailable vitamin D and vitamin D metabolite ratio (VMR) have emerged as potential novel vitamin D markers. We developed a multiplex liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine all elements necessary for the calculation of bioavailable vitamin D and VMR, including 25-hydroxyvitamin D [25-(OH)D] and 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3], VDBP and its isoforms, and albumin. Following separate reactions of hexane extraction and trypsin digestion, serum samples were analyzed using LC-MS/MS to measure 25-(OH)D3, 25-(OH)D2, 24,25-(OH)2D3, VDBP and its isoforms, and albumin. Analytical performances were assessed. Korean (n = 229), Arab (n = 98), White (n = 99) and Black American (n = 99) samples were analyzed. Bioavailable vitamin D and VMR were calculated. All target molecules were clearly separated and accurately quantified by LC-MS/MS. Analytical performances, including imprecision, accuracy, ion suppression, limit of quantification, linearity, and comparison with existing methods were within acceptable levels. The allele frequencies of VDBP isoforms in various races resulted similar to previously known values. The levels of bioavailable vitamin D were highest in White Americans and lowest in Black Americans. We have successfully developed a multiplex LC-MS/MS-based assay method that can simultaneously perform the measurement of all parameters needed to calculate bioavailable vitamin D and VMR. Our devised method was robust and reliable in terms of analytical performances and could be applied to routine clinical samples in the future to more accurately assess vitamin D status.

Evaluation of Vitamin D Metabolism in Patients with Type 1 Diabetes Mellitus in the Setting of Cholecalciferol Treatment.

In this prospective controlled study, we examined 25 adults with adequately controlled (HbA1c level < 8.0%) type 1 diabetes mellitus (T1DM) and 49 conditionally healthy adults, intending to reveal the diversity of vitamin D metabolism in the setting of cholecalciferol intake at a therapeutic dose. All patients received a single dose (150,000 IU) of cholecalciferol aqueous solution orally. Laboratory assessments including serum vitamin D metabolites (25(OH)D3, 25(OH)D2, 1,25(OH)2D3, 3-epi-25(OH)D3 and 24,25(OH)2D3), free 25(OH)D, vitamin D-binding protein (DBP) and parathyroid hormone (PTH) as well as serum and urine biochemical parameters were performed before the intake and on Days 1, 3 and 7 after the administration. The studied groups had no significant differences in baseline parameters except that the patients with diabetes showed higher baseline levels of free 25(OH)D (p < 0.05). They also lacked a correlation between the measured and calculated free 25(OH)D in contrast to the patients from the control group (r = 0.41, p > 0.05 vs. r = 0.88, p < 0.05), possibly due to the glycosylation of binding proteins, which affects the affinity constant for 25(OH)D. The elevation of vitamin D levels after the administration of cholecalciferol was comparable in both groups, with slightly higher 25(OH)D3 levels observed in the diabetes group throughout the study since Day 1 (p < 0.05). Overall, our data indicate that in patients with adequately controlled T1DM 25(OH)D3 levels and the therapeutic response to cholecalciferol is similar to that in healthy individuals.

Simultaneous determination of 24,25- and 25,26-dihydroxyvitamin D3 in serum samples with liquid-chromatography mass spectrometry - A useful tool for the assessment of vitamin D metabolism.

Vitamin D status is typically assessed by the measurement of 25-hydroxyvitamin D (25(OH)D). However, in selected patient groups the sole determination of 25(OH)D has been proven insufficient for this purpose. The simultaneous measurement of additional vitamin D metabolites may provide useful information for a better evaluation of the vitamin D status. Therefore, we developed and validated a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of 25(OH)D3, 25(OH)D2, 24,25(OH)2D3 and additionally 25,26(OH)2D3, which was identified with a synthesized pure substance. Pure and deuterated substances were used to prepare calibrators and internal standards for all target metabolites. Pre-analytical sample preparation comprised protein precipitation followed by liquid-liquid-extraction and derivatization with 4-Phenyl-1,2,4-triazole-3,5-dione (PTAD) using 50 µL sample volume. Samples were analyzed on an Agilent HPLC 1260 system equipped with a silica-based Kinetex® 5 µm F5 100 Å core-shell column (150 × 4.6 mm) coupled to a Sciex 4500 mass spectrometer. For all four metabolites, limit of detection (LoD) and limit of quantification (LoQ) ranged from 0.3 to 1.5 nmol/L and 1.0 to 3.1 nmol/L, respectively. Recovery varied between 76.1 % and 84.3 %. Intra- and inter-assay imprecision were <8.6 % and <11.5 %, respectively. The analysis of external and internal quality control samples showed good accuracy for 25(OH)D3, 25(OH)D2, 24(R),25(OH)2D3 and 25,26(OH)2D3. Method comparison studies with human samples that were also analyzed with two other LC-MS/MS methods showed close agreement. Finally, the present method has been shown capable of identifying patients with 24-hydroxylase deficiency, which proves its clinical utility.

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

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

Intake of ergosterol increases the vitamin D concentrations in serum and liver of mice.

Factors that can modify the bioavailability of orally administered vitamin D are not yet widely known. Ergosterol is a common fungal sterol found in food which has a chemical structure comparable to that of vitamin D. This study aimed to investigate the effect of ergosterol on vitamin D metabolism. Therefore, 36 male wild type-mice were randomly subdivided into three groups (n = 12) and received a diet containing 25 µg vitamin D3 and either 0 mg (control), 2 mg or 7 mg ergosterol per kg diet for 6 weeks. To elucidate the impact of ergosterol on hepatic hydroxylation of vitamin D, human hepatoma cells (HepG2) were treated with different concentrations of ergosterol. Concentrations of vitamin D3 and 25-hydroxyvitamin D3 (25(OH)D3) in cells, livers and kidneys of mice and additionally 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) in serum were quantified by LC-MS/MS. The concentration of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in serum was analyzed by commercially-available enzyme immuno assay. The concentrations of cholesterol and triglycerides were analyzed in livers of mice by photometric assays. Analyses revealed that mice receiving 7 mg/kg ergosterol with their diet had 1.3-, 1.7- and 1.5-times higher concentrations of vitamin D3 in serum, liver and kidney, respectively, than control mice (P < 0.05), whereas no significant effects were observed in mice fed 2 mg/kg ergosterol. The hydroxylation of vitamin D remained unaffected by dietary ergosterol, since the concentration of 25-hydroxyvitamin D3 in serum and tissues and the concentrations of 1,25(OH)2D3 and 24,25(OH)2D3 in serum were not different between the three groups of mice. The lipid concentrations in liver were also not affected by dietary ergosterol. Data from the cell culture studies showed that ergosterol did not influence the conversion of vitamin D3 to 25(OH)D3. To conclude, ergosterol appears to be a modulator of vitamin D3 concentrations in the body of mice, without modulating the hydroxylation of vitamin D3 in liver.

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

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

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.

Serum and synovial fluid vitamin D metabolites and rheumatoid arthritis.

Vitamin D-deficiency has been linked to inflammatory diseases including rheumatoid arthritis (RA). Studies to date have focused on the impact of serum 25-hydroxyvitamin D3 (25(OH)D3), an inactive form of vitamin D, on RA disease activity and progression. However, anti-inflammatory actions of vitamin D are likely to be mediated at sites of RA disease, namely the inflamed joint, and may involve other vitamin D metabolites notably the active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). In the current study serum and synovial fluid samples from n = 20 patients with persistent RA and n = 7 patients with reactive arthritis (ReA) were analysed for multiple vitamin D metabolites. Serum data for RA and ReA patients were compared to healthy controls (HC). There was no significant difference between RA or ReA patients relative to HC for 25(OH)D3, 24,25(OH)2D3, 1,25(OH)2D3 or 25(OH)D2. However, 3-epi-25(OH)D3 was significantly lower in RA and ReA patients compared to HC (p < 0.05). All vitamin D metabolites, apart from 25(OH)D2, were lower in SF compared to serum, and SF 1,25(OH)2D3 was unquantifiable in 13/20 RA and 4/7 ReA samples. SF 25(OH)D3, 3-epi-25(OH)D3 and DBP correlated inversely with swollen joint score, and serum 25(OH)D2 and SF DBP correlated directly with C-reactive protein levels. These data indicate that serum 25(OH)D3 provides only limited insight into the role of vitamin D in RA. Alternative serum metabolites such as 3-epi-25(OH)2D3, and SF metabolites, notably lack of SF 1,25(OH)2D3, may be more closely linked to RA disease severity and progress.

Rapid liquid chromatography-tandem mass spectrometry method for determination of 24,25(OH)2D and 25OHD with efficient separation of 3-epi analogs.

This study establishes and validates a rapid method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) without derivatization steps to simultaneously measure of 24,25(OH)2D2, 24,25(OH)2D3, 25OHD2, and 25OHD3, while efficiently separating the 3-epi analogs. Samples were prepared by precipitation and liquid-liquid extraction. The linearity, precision, accuracy, recovery and matrix effect of the method were thoroughly evaluated according to the Clinical & Laboratory Standards Institute guidelines. Additionally, the four vitamin D metabolites in the serum of 38 apparently healthy Chinese volunteers were evaluated. The total analysis time was 8.0 min, with efficient separation of 3-epi 24,25(OH)2D3 and 3-epi 25OHD3, without interference from isomers such as 23,25(OH)2D3 or 1,25(OH)2D2, 1,25(OH)2D3. Good reproducibility was obtained for all four metabolites with within-run coefficient variations (CVs) of 4.07%-6.55%, 4.26%-7.84%, 2.46%-7.21%, and 4.90%-6.87% for 25OHD3, 25OHD2, 24,25(OH)2D3, and 24,25(OH)2D2, respectively, and the total CVs were 4.29%-6.64%, 6.14%-7.84%, 4.33%-7.21%, 5.82%-9.90%, respectively. The limit of quantification was 0.625 ng/mL for 25OHD3 and 25OHD2, and 0.5 ng/mL for 24,25(OH)2D3 and 24,25(OH)2D2. The relative bias of the LC-MS/MS method compared to the certified results of SRM 972a for 25OHD3, 25OHD2 and 24,25(OH)2D3 was -2.21% to 1.01%, 3.38% to 6.73%, and -7.72% to -3.9%, respectively. The mean±SD values for 25OHD, 24,25(OH)2D and 25OHD/24,25(OH)2D in the volunteers were 13.5±4.4 ng/mL(range:7.6-27.5 ng/mL), 0.84±0.42 ng/mL (range:0.26-2.1 ng/mL), and 18±7(range:8-37), respectively. Thus, a simple, precise LC-MS/MS method for appropriate retention and separation of vitamin D metabolites and their epi analogs was developed.

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.

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

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

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.

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.

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.

Activation of Reactive MALDI Adduct Ions Enables Differentiation of Dihydroxylated Vitamin D Isomers.

Vitamin D compounds are secosteroids, which are best known for their role in bone health. More recent studies have shown that vitamin D metabolites and catabolites such as dihydroxylated species (e.g., 1,25- and 24,25-dihydroxyvitamin D3) play key roles in the pathologies of various diseases. Identification of these isomers by mass spectrometry is challenging and currently relies on liquid chromatography, as the isomers exhibit virtually identical product ion spectra under collision induced dissociation conditions. Here, we developed a simple MALDI-CID method that utilizes ion activation of reactive analyte/matrix adducts to distinguish isomeric dihydroxyvitamin D3 species, without the need for chromatography separation or chemical derivatization techniques. Specifically, reactive 1,5-diaminonaphthalene adducts of dihydroxyvitamin D3 compounds formed during MADI were activated and specific cleavages in the secosteroid's backbone structure were achieved that produced isomer-diagnostic fragment ions. Graphical Abstract ᅟ.

Interlaboratory Comparison for the Determination of 24,25-Dihydroxyvitamin D3 in Human Serum Using Liquid Chromatography with Tandem Mass Spectrometry.

Six laboratories associated with the Vitamin D Standardization Program (VDSP) participated in an interlaboratory comparison of LC with tandem MS (MS/MS) methods for the determination of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] in human serum. The laboratories analyzed two different serum-based Standard Reference Materials (SRMs) intended for use in the determination of 25-hydroxyvitamin D and 30 samples from the Vitamin D External Quality Assessment Scheme (DEQAS). All laboratory methods for 24,25(OH)2D3 were based on isotope dilution LC-MS/MS; three of the methods used derivatization of the vitamin D metabolites before LC-MS/MS. Laboratory results were compared to the National Institute of Standards and Technology (NIST) results, which were obtained using their newly developed candidate reference measurement procedure for 24,25(OH)2D3. Laboratory results for the SRM samples varied in comparability to the NIST results, with one laboratory in excellent agreement (-1.6% mean bias), three laboratories at 10-15% mean bias, and the remaining laboratory at 36% mean bias. For the 30 DEQAS samples, the mean bias for the five laboratories ranged from 6 to 15%; however, the SD of the bias ranged from 8 to 29%. As a result of this intercomparison study, one laboratory discovered and corrected a method calculation error and another laboratory modified and improved their LC-MS/MS method.

Improved accuracy of an tandem liquid chromatography-mass spectrometry method measuring 24R,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D metabolites in serum using unspiked controls and its application to determining cross-reactivity of a chemiluminescent microparticle immunoassay.

Measurement of serum 25-hydroxyvitamin D [25(OH)D] is considered the best indicator of vitamin D status. Two minor vitamin D metabolites are common interferences encountered in 25(OH)D assays. The first is 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3], which if not chromatographically resolved from 25-hydroxyvitamin D3 [25(OH)D3], can overestimate 25(OH)D concentrations. The second is 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3], which can cross-react with the antibodies in 25(OH)D immunoassays. Our aim was to develop an LC-MS/MS method capable of detecting both 3-epi-25(OH)D3 and 24R,25(OH)2D3 in serum without the use of a derivatization agent. We report an isotope dilution LC-MS/MS method, with electrospray ionization in the positive mode, that can simultaneously detect 24R,25(OH)2D3, 25(OH)D3, 3-epi-25(OH)D3, and 25-hydroxyvitamin D2. The method employs a cost-effective liquid-liquid extraction using only 150µL of sera and a total run time of 10min. Method performance was assessed by using quality controls made from pooled sera as an alternative to sera spiked with analytes. Biobanked samples, originally analyzed by chemiluminescent microparticle immunoassay (CMIA), were re-analyzed with this method to determine the contribution of 24R,25(OH)2D3 cross-reactivity to 25(OH)D measurement bias. The CMIA over-estimation of 25(OH)D measurements relative to LC-MS/MS was found to depend on both 25(OH)D and 24R,25(OH)2D3 concentrations.