RESUMO
Vitamin D analysis by MS faces several analytical challenges, including inefficient ionization, nonspecific fragmentation, interferences from epimers, isomers, and isobars, as well as very low concentration levels. In this study, we used 2-fluoro-1-methylpyridinium (FMP) p-toluene sulfonate for derivatization of vitamin D3 metabolites to increase detection sensitivity and allow for full chromatographic separation of vitamin D isomers and epimers. UHPLC-MS/MS was used for measurement of five vitamin D3 metabolites in human serum. Compared with Amplifex and 4-phenyl-1,2,4-triazolin-3,5-dion, the FMP p-toluene sulfonate reaction required less time to be performed. The method was optimized and validated to ensure accuracy, precision, and reliability. In-house and commercial quality control samples were used to assure the quality of the results for 25-hydroxyvitamin D3. The method showed very good linearity and intraday and interday accuracy and precision; coefficients of determination (r2) ranged between 0.9977 and 0.9992, relative recovery from 95 to 111%, and coefficient of variation from 0.9 to 11.3. Stability tests showed that the extracted derivatized serum samples were stable for 24 h after storage at -20°C; 24,25-dihydroxyvitamin D3 and 1,25-dihydroxyvitamin D3-FMP derivatives were stable for 1 week at -80°C. The method was applied to samples of healthy individuals for quantitative determination of vitamin D3, the two epimers of 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3.
Assuntos
Calcifediol , Espectrometria de Massas em Tandem , Humanos , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Tolueno , Reprodutibilidade dos Testes , 24,25-Di-Hidroxivitamina D 3 , Vitamina D , Vitaminas , ColecalciferolRESUMO
Phosphate (P) is an essential element involved in various biological actions, such as bone integrity, energy production, cell signaling and molecular component. P homeostasis is modulated by 4 main tissues; intestine, kidney, bone, and parathyroid gland, where 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), parathyroid hormone and fibroblast growth factor 23 (FGF23) are produced and/or have an influence. In bone, serum P level modulates the production of FGF23 which then controls not only P excretion but also vitamin D metabolism in kidney in an endocrine manner. The hormonally active form of vitamin D, 1,25(OH)2D3, also has a significant effect on skeletal cells via its receptor, the vitamin D receptor, to control gene expression which mediates bone metabolism as well as mineral homeostasis. In this study, we adopted RNA-seq analysis to understand genome-wide skeletal gene expression regulation in response to P and 1,25(OH)2D3. We examined lumbar 5 vertebrae from the mice that were fed P deficient diet for a week followed by an acute high P diet for 3, 6, and 24 h as well as mice treated with 1,25(OH)2D3 intraperitoneally for 6 h. Further identification and exploration of the genes regulated by P and 1,25(OH)2D3 showed that P dynamically modulates the expression of skeletal genes involved in various biological processes while 1,25(OH)2D3 regulates genes highly related to bone metabolism. Our in vivo data were then compared with in vitro data that we previously obtained, which suggests that the gene expression profiles presented in this report mainly represent those of osteocytes. Interestingly, it was found that even though the skeletal response to P is distinguished from that to 1,25(OH)2D3, both factors have an effect on Wnt signaling pathway to modulate bone homeostasis. Taken together, this report presents genome-wide data that provide a foundation to understand molecular mechanisms by which skeletal cells respond to P and 1,25(OH)2D3.
Assuntos
Calcitriol , Fosfatos , Camundongos , Animais , Calcitriol/farmacologia , Calcitriol/metabolismo , Transcriptoma , Estudo de Associação Genômica Ampla , Vitamina D/farmacologia , Vitamina D/metabolismo , 24,25-Di-Hidroxivitamina D 3 , Cálcio/metabolismoRESUMO
Ninety archived human serum samples from the Vitamin D External Quality Assessment Scheme (DEQAS) were analyzed using a reference measurement procedure (RMP) based on isotope dilution liquid chromatography - tandem mass spectrometry (ID LC-MS/MS) for the determination of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3]. These 24,25(OH)2D3 results, in conjunction with concentration values assigned using RMPs for 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3], provide a valuable resource for assessing the accuracy of measurements for 24,25(OH)2D3 and for investigating the relationship between 24,25(OH)2D3 and 25(OH)D3. Results for 24,25(OH)2D3 using the RMP were compared to DEQAS consensus values demonstrating that the consensus values were not sufficient to assess the accuracy of measurements among different laboratories and methods. A multivariable regression analysis approach using historical DEQAS consensus values for various total 25(OH)D assays was used to assess the contribution of 24,25(OH)2D3 concentration on the assay response. The response of several ligand binding assays for total 25(OH)D was shown to be impacted by the presence of 24,25(OH)2D3.
Assuntos
Espectrometria de Massas em Tandem , Vitamina D , Humanos , Cromatografia Líquida , Espectrometria de Massas em Tandem/métodos , Vitaminas , Calcifediol , 24,25-Di-Hidroxivitamina D 3RESUMO
Patients with cystic fibrosis (CF) commonly have lower circulating concentrations of 25-hydroxyvitamin D (25(OH)D) than healthy populations. We comprehensively compared measures of vitamin D metabolism among individuals with CF and healthy control subjects. In a cross-sectional study, serum from participants with CF (N = 83) and frequency-matched healthy control subjects by age and race (N = 82) were analyzed for: 25(OH)D2 and 25(OH)D3, 1α,25-dihydroxyvitamins D2 and D3 (1α,25(OH)2D2 and 1α,25(OH)2D3), 24,25-dihydroxyvitamin D3 (24,25(OH)2D3), 4ß,25-dihydroxyvitamin D3 (4ß,25(OH)2D3), 25-hydroxyvitamin D3-3-sulfate (25(OH)D3-S), and 25-hydroxyvitamin D3-3-glucuronide (25(OH)D3-G). In a 56-day prospective pharmacokinetic study, â¼25 µg deuterium-labeled 25(OH)D3 (d6-25(OH)D3) was administered intravenously to participants (N = 5 with CF, N = 5 control subjects). Serum was analyzed for d6-25(OH)D3 and d6-24,25(OH)2D3, and pharmacokinetic parameters were estimated. In the cross-sectional study, participants with CF had similar mean (SD) total 25(OH)D concentrations as control subjects (26.7 [12.3] vs. 27.7 [9.9] ng/mL) and had higher vitamin D supplement use (53% vs. 22%). However, participants with CF had lower total 1α,25(OH)2D (43.6 [12.7] vs. 50.7 [13.0] pg/mL), 4ß,25(OH)2D3 (52.1 [38.9] vs. 79.9 [60.2] pg/mL), and 25(OH)D3-S (17.7 [11.6] vs. 30.1 [12.3] ng/mL) (p < 0.001 for all). The pharmacokinetics of d6-25(OH)D3 and d6-24,25(OH)D3 did not differ between groups. In summary, although 25(OH)D concentrations were comparable, participants with CF had lower 1α,25(OH)2D, 4ß,25(OH)2D3, and 25(OH)D3-S concentrations than healthy controls. Neither 25(OH)D3 clearance, nor formation of 24,25(OH)2D3, appears to account for these differences and alternative mechanisms for low 25(OH)D in CF (i.e., decreased formation, altered enterohepatic recirculation) should be explored.
Assuntos
Fibrose Cística , Humanos , Estudos Prospectivos , Estudos Transversais , Vitaminas/farmacocinética , Vitamina D , Calcifediol , 24,25-Di-Hidroxivitamina D 3RESUMO
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
Assuntos
Humanos , Masculino , Feminino , Idoso , Idoso de 80 Anos ou mais , Deficiência de Vitamina D/diagnóstico , 24,25-Di-Hidroxivitamina D 3/sangue , Avaliação Geriátrica , Saúde do Idoso Institucionalizado , Estudos TransversaisRESUMO
The complete understanding of the excretion of surplus 25-hydroxyvitamin D<sub>3</sub> [25(OH)D<sub>3</sub>] in humans remains to be accomplished. In our previous study, 24,25-dihydroxyvitamin D<sub>3</sub> [24,25(OH)<sub>2</sub>D<sub>3</sub>] 24-glucuronide was identified as a major urinary vitamin D<sub>3</sub> metabolite, while the glucuronide of 23,25-dihydroxyvitamin D<sub>3</sub> [23,25(OH)<sub>2</sub>D<sub>3</sub>] is another metabolite of interest but has not been sufficiently evaluated. Although the quantitative analysis of 24,25(OH)<sub>2</sub>D<sub>3</sub> liberated in urine by the treatment with ß-glucuronidase (GUS) has been conducted, no information was provided about the amount of the glucuronidated 23,25(OH)<sub>2</sub>D<sub>3</sub> in the urine. In this study, we first developed and validated a liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS)-based method for the simultaneous quantification of 23,25(OH)<sub>2</sub>D<sub>3</sub> and 24,25(OH)<sub>2</sub>D<sub>3</sub> liberated in urine by GUS. The analysis of the urine samples revealed that the amount of 23,25(OH)<sub>2</sub>D<sub>3</sub> was almost as much as that of 24,25(OH)<sub>2</sub>D<sub>3</sub>, in contrast to the fact that the plasma concentration of 23,25(OH)<sub>2</sub>D<sub>3</sub> was much lower than that of 24,25(OH)<sub>2</sub>D<sub>3</sub>. These results strongly suggested that 23,25(OH)<sub>2</sub>D<sub>3</sub> is more susceptible to glucuronidation and more promptly excreted into urine than 24,25(OH)<sub>2</sub>D<sub>3</sub>. Furthermore, the amount ratios of 23,25(OH)<sub>2</sub>D<sub>3</sub> to 24,25(OH)<sub>2</sub>D<sub>3</sub> in the urine samples did not markedly vary during the day (morning/evening) and even by a week-long vitamin D<sub>3</sub> supplementation (1000 IU/body/day). We concluded that the C-23 hydroxylation plays a crucial role in the urinary excretion of surplus 25(OH)D<sub>3</sub>.
Assuntos
Colecalciferol , Espectrometria de Massas em Tandem , 24,25-Di-Hidroxivitamina D 3 , Cromatografia Líquida/métodos , Glucuronidase , Glucuronídeos , Humanos , Espectrometria de Massas em Tandem/métodos , Vitamina D/análogos & derivadosRESUMO
Low 25-hydroxyvitamin D (25(OH)D) concentration is a recognised risk factor for multiple sclerosis (MS). Associations with vitamin D metabolites and vitamin D binding globulin (VDBG) have not been widely studied. We assessed the association between vitamin D metabolites (25(OH)D2, 25(OH)D3, c3-epimer 25(OH)D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3)) measured by liquid chromatography-tandem mass spectrometry assays, VDBG measured using a polyclonal immunoassay, and calculated free and bioavailable 25(OH)D, free 1,25(OH)2D3, and the 24,25(OH)2D3: total 25(OH)D and total 1,25(OH)2D: total 25(OH)D ratios with risk of a first clinical diagnosis of CNS demyelination (FCD) in an Australian case-control study (n = 196 cases, n = 241 controls, matched on age, sex and study region). Higher 25(OH)D (adjusted odds ratio (AOR) = 0.94 (95 % confidence interval (CI) 0.85-1.03) per 10 nmol/L increment) and 24,25(OH)2D3 (AOR = 0.81 (95 %CI 0.65-1.00) per 1 nmol/L increment) concentrations were associated with reduced FCD risk. Our results were compatible with no association for the other vitamin D metabolites, ratios, or VDBG with FCD risk. Thus, using standardised assays, and a comprehensive range of vitamin D metabolites, we confirmed the association of higher 25(OH)D and reduced FCD risk, and describe a similar effect for 24,25(OH)2D3; free or bioavailable 25(OH)D were not associated with FCD risk.
Assuntos
Doenças Desmielinizantes , Espectrometria de Massas em Tandem , 24,25-Di-Hidroxivitamina D 3 , Austrália , Estudos de Casos e Controles , Sistema Nervoso Central , Doenças Desmielinizantes/diagnóstico , Ergocalciferóis , Humanos , Espectrometria de Massas em Tandem/métodos , Vitamina D , Proteína de Ligação a Vitamina DRESUMO
The formation of 24,25-dihydroxyvitamin D (24,25(OH)2D) from 25-hydroxyvitamin D (25(OH)D) is the primary mechanism for the metabolic clearance of 25(OH)D, and is regulated by tissue-level vitamin D activity. The ratio of 24,25(OH)2D3 to 25(OH)D3 in blood (vitamin D metabolite ratio, VDMR) is postulated to be a marker of 25(OH)D3 clearance, however this has never been tested. We measured baseline 24,25(OH)2D3 and 25(OH)D3 concentrations in 87 participants by liquid chromatography-tandem mass spectrometry. Following an infusion of deuterated 25(OH)D3, blood samples for each participant were collected over 56 days and analyzed for deuterated vitamin D metabolites. 25(OH)D3 clearance and the deuterated metabolite-to-parent AUC ratio (ratio of the AUC of deuterated 24,25(OH)2D3 to that of deuterated 25(OH)D3) were calculated. We compared the VDMR with these two measures using correlation coefficients and linear regression. Participants had a mean age of 64 ± 11years, 41 % were female, 30 % were self-described Black, 28 % had non-dialysis chronic kidney disease (CKD) and 23 % had kidney failure treated with hemodialysis. The VDMR was strongly correlated with 25(OH)D3 clearance and the deuterated metabolite-to-parent AUC ratio (r = 0.51 and 0.76, respectively). Adjusting for 25(OH)D3 clearance or the deuterated metabolite-to-parent AUC ratio in addition to clinical covariates, lower VDMR was observed in participants with CKD and kidney failure than in healthy controls; in Black than White participants; and in those with lower serum albumin. Our findings validate the VDMR as a measure of 25(OH)D3 clearance. This relationship was biased by characteristics including race and kidney disease, which warrant consideration in studies assessing the VDMR.
Assuntos
Calcifediol , Insuficiência Renal Crônica , 24,25-Di-Hidroxivitamina D 3 , Idoso , Biomarcadores , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Vitamina D/análogos & derivados , VitaminasRESUMO
OBJECTIVES: In-house developed liquid-chromatography mass spectrometry (LC-MS/MS) methods are used more and more frequently for the simultaneous quantification of vitamin D metabolites. Among these, 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) is of clinical interest. This study assessed the agreement of this metabolite in two validated in-house LC-MS/MS methods. METHODS: 24,25(OH)2D3 was measured in 20 samples from the vitamin D external quality assurance (DEQAS) program and in a mixed cohort of hospital patients samples (n=195) with the LC-MS/MS method at the Medical University of Graz (LC-MS/MS 1) and at the University of Liège (LC-MS/MS 2). RESULTS: In DEQAS samples, 24,25(OH)2D3 results with LC-MS/MS 1 had a proportional bias of 1.0% and a negative systemic difference of -0.05%. LC-MS/MS 2 also showed a proportional bias of 1.0% and the negative systemic bias was -0.22%. Comparing the EQA samples with both methods, no systemic bias was found (0.0%) and the slope was 1%. The mean difference of 195 serum sample measurements between the two LC-MS/MS methods was minimal (-0.2%). Both LC-MS/MS methods showed a constant bias of 0.31 nmol/L and a positive proportional bias of 0.90%, respectively. CONCLUSIONS: This study is the first to assess the comparability of 24,25(OH)2D3 concentrations in a mixed cohort of hospitalized patients with two fully validated in-house LC-MS/MS methods. Despite different sample preparation, chromatographic separation and ionization, both methods showed high precision measurements of 24,25(OH)2D3. Furthermore, we demonstrate the improvement of accuracy and precision measurements of 24,25(OH)2D3 in serum samples and in the DEQAS program.
Assuntos
Espectrometria de Massas em Tandem , Vitamina D , 24,25-Di-Hidroxivitamina D 3 , Cromatografia Líquida/métodos , Humanos , Manejo de Espécimes , Espectrometria de Massas em Tandem/métodosRESUMO
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.
Assuntos
24,25-Di-Hidroxivitamina D 3/sangue , Proteína de Ligação a Vitamina D/sangue , Vitamina D/análogos & derivados , Vitamina D/genética , 24,25-Di-Hidroxivitamina D 3/isolamento & purificação , Disponibilidade Biológica , Calcifediol/farmacologia , Cromatografia Líquida , Humanos , Isoformas de Proteínas/sangue , Isoformas de Proteínas/isolamento & purificação , Albumina Sérica/isolamento & purificação , Espectrometria de Massas em Tandem , Vitamina D/sangue , Vitamina D/isolamento & purificação , Vitamina D/metabolismo , Proteína de Ligação a Vitamina D/isolamento & purificaçãoRESUMO
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.
Assuntos
Colecalciferol/administração & dosagem , Diabetes Mellitus Tipo 1/sangue , Vitamina D/sangue , 24,25-Di-Hidroxivitamina D 3/sangue , 25-Hidroxivitamina D 2/sangue , Administração Oral , Adulto , Calcifediol/sangue , Calcitriol/sangue , Estudos de Casos e Controles , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/urina , Feminino , Hemoglobinas Glicadas/análise , Humanos , Masculino , Hormônio Paratireóideo/sangue , Estudos Prospectivos , Vitamina D/metabolismo , Proteína de Ligação a Vitamina D/sangue , Adulto JovemRESUMO
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.
Assuntos
24,25-Di-Hidroxivitamina D 3/sangue , Cromatografia Líquida/métodos , Di-Hidroxicolecalciferóis/sangue , Espectrometria de Massas em Tandem/métodos , Humanos , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes , Vitamina D/metabolismoRESUMO
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.
Assuntos
24,25-Di-Hidroxivitamina D 3/sangue , Calcifediol/sangue , Calcitriol/sangue , Callithrix/metabolismo , Fatores Etários , Animais , Animais de Zoológico/metabolismo , Colecalciferol/sangue , Feminino , Masculino , Valores de Referência , Fatores SexuaisRESUMO
Background Simultaneous measurement of 25(OH)D and 24,25(OH)2D is a new tool for predicting vitamin D deficiency and allows evaluating CYP24A1 lack of function. Interpretation of 24,25(OH)2D should be performed according to 25(OH)D levels and a ratio, called the vitamin D metabolite ratio (VMR) has been proposed for such a purpose. Unfortunately, the VMR can be expressed in different ways and cannot be used if 24,25(OH)2D concentrations are undetectable. Here, we propose evaluating the enzyme activity taking into consideration the probability that a normal population presents undetectable 24,25(OH)2D concentrations according to 25(OH)D levels. We thus retrospectively measured 25(OH)D and 24,25(OH)2D in a population of 1200 young subjects to evaluate the 25(OH)D threshold above which the enzyme was induced. Methods Serum samples from 1200 infants, children, adolescent and young adults were used to simultaneously quantify 25(OH)D and 24,25(OH)2D by LCMS/MS. Results Median (interquartile range [IQR]) levels were 20.6 (14.4-27.2) ng/mL for 25(OH)D. 172 subjects (14.3%) presented 24,25(OH)2D values below the LOQ. When 25(OH)D values were <11 ng/mL, 63.1% of subjects presented undetectable 24,25(OH)2D concentrations. Percentage decreased with increasing 25(OH)D values to become 19.7% for 25(OH)D comprised between 12 and 15 ng/mL, 5.1% for 25(OH)D between 16 and 20 and 0.7% for 25(OH)D >21 ng/mL. Conclusions We suggest using a statistical approach to evaluate CYP24A1 function according to 25(OH)D concentrations. Our results also show that vitamin D deficiency, as defined biochemically, could be around 20 ng/mL in infants, children, adolescent and young adults and that vitamin D deficiency could be evaluated on a more individual basis.
Assuntos
24,25-Di-Hidroxivitamina D 3/análise , Calcifediol/análise , Deficiência de Vitamina D/patologia , Vitamina D3 24-Hidroxilase/genética , Adolescente , Criança , Pré-Escolar , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Lactente , Limite de Detecção , Masculino , Polimorfismo de Nucleotídeo Único , Estudos Retrospectivos , Espectrometria de Massas em Tandem , Deficiência de Vitamina D/genética , Adulto JovemRESUMO
BACKGROUND Vitamin D3 (VD3) is a commonly used supplement in clinical practice. Cytochrome P450 3A11 (CYP3A11) is the most important monomeric enzyme involved in metabolism of drugs. This study aimed to investigate effects of vitamin D3 (VD3) on CYP3A11 activity. MATERIAL AND METHODS Forty male Sprague-Dawley (SD) rats were randomly divided a Control group (peanut oil 0.1 ml/kg/d), a Low-VD3 group (100 IU/kg/d), a Medium-VD3 group (400 IU/kg/d), and a High-VD3 (1600 IU/kg/d) group. Blood samples were collected from the jugular vein after midazolam (MDZ) administration. CYP3A11 expressions in liver and colon were detected by Western blotting and immunohistochemistry (IHC) assay. The concentration of serum 25(OH)D3 and serum 1,25(OH)2D3 were evaluated using ELISA. Effects of different dosages of vitamin D3 on metabolism of MDZ were evaluated using high-performance liquid chromatography (HPLC). RESULTS Vitamin D3 significantly enhanced serum 25(OH)D3 and 1,25(OH)2D3 levels in rats compared to Control rats (p<0.05). Expressions of hepatic CYP3A11 were more than 10-fold higher in rats treated with vitamin D3 compared to Control rats (p<0.05). Expressions of colon CYP3A11 were 5-fold higher than in Control rats (p<0.05). CYP3A11 expressions in vitamin D3-treated groups were significantly higher compared to the Control group (p<0.05). MDZ levels were significantly higher in Vitamin D3-treated rats compared to that in Control rats (p<0.05). Concentrations of serum MDZ at every sampling point were remarkably lower in the vitamin D3-treated rats than in Control rats (p<0.05). CONCLUSIONS Vitamin D3 was transformed into 1,25(OH)2D3 by triggering CYP3A11 and CYP3A11 activity and by hydrolyzing MDZ.
Assuntos
Colecalciferol/metabolismo , Citocromo P-450 CYP3A/metabolismo , 24,25-Di-Hidroxivitamina D 3/metabolismo , Animais , Calcifediol , Calcitriol/farmacologia , China , Cromatografia Líquida de Alta Pressão/métodos , Citocromo P-450 CYP3A/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Masculino , Midazolam/metabolismo , Ratos , Ratos Sprague-Dawley , Vitamina D/sangue , VitaminasRESUMO
Vitamin D is a fat-soluble prohormone that can be activated both systemically and within individual tissues. Our lab has previously demonstrated that the corneal epithelium can activate vitamin D and that the vitamin D metabolites 1,25(OH)2D3 and 24R,25(OH)2D3 can affect corneal epithelial migration, proliferation, and tight and gap junction function. These vitamin D-derived metabolites signal through the vitamin D receptor (VDR). The purpose of this study was to specifically determine the effects of 1,25(OH)2D3 and 24R,25(OH)2D3 on corneal epithelial cell gap junction proteins. Connexin (Cx) 26, 30 and 43 protein expression was detected in a human corneal epithelial cell line (HCEC), wild type and vitamin D receptor knockout (VDR-/-) mouse corneas, and cultured mouse primary epithelial cells (MPCEC). In vitro gap junction function was assessed using the scrape loading/dye transfer assay. HCEC and MPCEC were treated with 1,25(OH)2D3 or 24R,25(OH)2D3. Western blotting was used to detect gap junction proteins. Vitamin D3 effects on epithelial intracellular Ca++ (Ca++i) were determined using the dye Cal-520. Cx26 and Cx43 protein levels were significantly increased in HCEC and MPCEC treated with both 1,25(OH)2D3 and 24R,25(OH)2D3. Cx30 and Cx43 protein levels were also significantly increased in VDR-/- MPCEC. In vitro gap junction connectivity was significanlty enhanced in HCEC and MPCEC cultured with 24R,25(OH)2D3 and 1,25(OH)2D3. Ca++i was not affected by 1,25(OH)2D3 or 24R,25(OH)2D3 in HCEC or MPCEC. We conclude that both 1,25(OH)2D3 and 24R,25(OH)2D3 are positive regulators of connexin proteins and gap junction communication in the corneal epithelium. These vitamin D metabolites appear to signal through both VDR-dependent and -independent pathways. The effects of vitamin D on corneal epithelial gap junctions do not seem to be dependent on Ca++i.
Assuntos
24,25-Di-Hidroxivitamina D 3/farmacologia , Calcitriol/farmacologia , Conexinas/metabolismo , Epitélio Corneano/efeitos dos fármacos , Receptores de Calcitriol/fisiologia , Animais , Western Blotting , Cálcio/metabolismo , Linhagem Celular , Conexina 26 , Conexina 30/metabolismo , Conexina 43/metabolismo , Epitélio Corneano/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de SinaisRESUMO
Vitamin D has long been prescribed as a supplement to breast cancer patients. This is partially motivated by data indicating that low serum vitamin D, measured as 25-hydroxyvitamin D3 [25(OH)D3], is associated with worsened cancer prognosis and decreased survival rates in cancer patients. However, clinical studies investigating the role of vitamin D supplementation in breast cancer treatment are largely inconclusive. One reason for this may be that many of these studies ignore the complexity of the vitamin D metabolome and the effects of these metabolites at the cellular level. Once ingested, vitamin D is metabolized into 37 different metabolites, including 25(OH)D3, which is the metabolite actually measured clinically, as well as 1,25(OH)2D3 and 24,25(OH)2D3. Recent work by our lab and others has demonstrated a role for 24R,25(OH)2D3, in the modulation of breast cancer tumors via an estrogen receptor α-dependent mechanism. This review highlights the importance of considering estrogen receptor status in vitamin d-associated prognostic studies of breast cancer and proposes a potential mechanism for 24R,25(OH)2D3 signaling in breast cancer cells.
Assuntos
24,25-Di-Hidroxivitamina D 3/farmacologia , Neoplasias da Mama/dietoterapia , Neoplasias da Mama/patologia , Receptores de Estrogênio/metabolismo , 24,25-Di-Hidroxivitamina D 3/metabolismo , Animais , Neoplasias da Mama/metabolismo , Feminino , Humanos , Neoplasias Mamárias Experimentais/dietoterapia , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologiaRESUMO
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 (nâ¯=â¯12) and received a diet containing 25⯵g vitamin D3 and either 0â¯mg (control), 2â¯mg or 7â¯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 7â¯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 2â¯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.
Assuntos
Colecalciferol/farmacologia , Ergosterol/farmacologia , Vitaminas/farmacologia , 24,25-Di-Hidroxivitamina D 3/sangue , 24,25-Di-Hidroxivitamina D 3/metabolismo , Animais , Calcifediol/sangue , Calcifediol/metabolismo , Colecalciferol/sangue , Colecalciferol/farmacocinética , Células Hep G2 , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Vitaminas/sangue , Vitaminas/farmacocinéticaRESUMO
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.
Assuntos
Colecalciferol/farmacologia , Suplementos Nutricionais , Ergocalciferóis/farmacologia , Vitaminas/farmacologia , 24,25-Di-Hidroxivitamina D 3/sangue , 25-Hidroxivitamina D 2/sangue , Adulto , Idoso , Calcifediol/sangue , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
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.