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1.
J Steroid Biochem Mol Biol ; 187: 27-33, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30389627

RESUMO

Two novel 20-hydroxyvitamin D3 analogues (4a,b) with the A-ring modification have been synthesized by a convergent manner. An alternative pathway of vitamin D3 metabolism by cytochrome P450scc CYP11A1 was reported to afford 20-hydroxyvitamin D3 (3), functions of which remain to be explored. Based on the structure of the 20-hydroxy metabolite, novel analogues (4a,b) with the modifications, including the 1α-hydroxy, 25-hydroxy and 2α-methyl groups, have been designed. The side chain of the requisite CD-ring portions (9a,b) was introduced by Grignard reaction as a key step, and the stereochemistry at the C20 position was confirmed by the X-ray crystal structure analysis of the synthetic intermediate (8b). Preliminary biological characterization using the bovine thymus vitamin D receptor suggested that the introduction of the active motifs into the 20-hydroxyvitamin D3 scaffold elevated the receptor affinity.


Assuntos
Calcifediol/análogos & derivados , Vitaminas/síntese química , Animais , Calcifediol/síntese química , Calcifediol/química , Calcifediol/farmacologia , Bovinos , Cristalografia por Raios X , Modelos Moleculares , Receptores de Calcitriol/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade , Vitaminas/química , Vitaminas/farmacologia
2.
J Steroid Biochem Mol Biol ; 186: 161-168, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30367940

RESUMO

Three 23-hydroxylated vitamin D3 derivatives, which are metabolites of 25-hydroxyvitamin D3 produced by CYP24A1 and a related diastereomer, were efficiently synthesized. Each C23 hydroxy unit was constructed by the Claisen condensation reaction with ethyl acetate or the Grignard reaction with 2-methylallymagnesium chloride. Stereochemistry at the C23 position was determined by a modified Mosher's method. The triene structures were constructed by the Wittig-Horner reaction utilizing the A-ring phosphine oxide moiety.


Assuntos
Calcifediol/metabolismo , Di-Hidroxicolecalciferóis/síntese química , Hidroxicolecalciferóis/síntese química , Calcifediol/análogos & derivados , Técnicas de Química Sintética , Di-Hidroxicolecalciferóis/química , Hidroxicolecalciferóis/química , Estereoisomerismo , Vitamina D3 24-Hidroxilase/metabolismo
3.
Sci Rep ; 8(1): 1478, 2018 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-29367669

RESUMO

20S-hydroxyvitamin D3 [20S(OH)D3] is anti-inflammatory and not hypercalcemic, suggesting its potential as a lead compound. In this study, side chain modified 20S(OH)D3 analogs (4, 13, 23 and 33) together with their 1α-OH derivatives were synthesized and their metabolism and biological activities tested. 4, 13 and 23 are good substrates for CYP27B1, enabling enzymatic synthesis of their 1α-OH derivatives 5, 14 and 24. However, 33 could not be hydroxylated by CYP27B1 and acts as an inhibitor. All analogs were poorer substrates for CYP24A1 than calcitriol, indicating improved catabolic stability. While the parent analogs showed minimal VDR stimulating activity, their 1α-OH derivatives were potent VDR agonists. 4, 5, 14 and 24 significantly upregulated the expression of CYP24A1 at the mRNA level, consistent with their VDR activation abilities and indicating that 1α-hydroxylation is required to produce analogs with strong activity. These analogs have anti-inflammatory activities that are influenced by side chain composition and by 1α-hydroxylation. To understand their molecular interactions with the VDR, 20S(OH)D3, 4 and 33 were co-crystalized with the VDR ligand binding domain, which revealed subtle differences to the calcitriol-bound receptor. This study demonstrates the potential of the 20S(OH)D3 scaffold for the development of novel anti-inflammatory agents.


Assuntos
Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Calcifediol/análogos & derivados , Proliferação de Células/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Receptores de Calcitriol/agonistas , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Calcifediol/química , Calcifediol/farmacologia , Células Cultivadas , Humanos , Hidroxilação , Queratinócitos/citologia , Queratinócitos/metabolismo , Vitamina D3 24-Hidroxilase/metabolismo
4.
J Steroid Biochem Mol Biol ; 177: 59-69, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28716760

RESUMO

Recent studies indicate that CYP2R1 is the major 25-hydroxylase catalyzing the first step in vitamin D activation. Since the catalytic properties of CYP2R1 have been poorly studied to date and it is a membrane protein, we examined the purified enzyme in a membrane environment. CYP2R1 was expressed in E. coli and purified by nickel affinity- and hydrophobic interaction-chromatography and assayed in a reconstituted membrane system comprising phospholipid vesicles plus purified human NADPH-P450 oxidoreductase. CYP2R1 converted vitamin D3 in the vesicle membrane to 25-hydroxyvitamin D3 [25(OH)D3] with good adherence to Michaelis-Menten kinetics. The kinetic parameters for 25-hydroxylation of vitamin D3 by the two major vitamin D 25-hydroxylases, CYP2R1 and CYP27A1, were examined in vesicles under identical conditions. CYP2R1 displayed a slightly lower kcat than CYP27A1 but a much lower Km for vitamin D3, and thus an overall 17-fold higher catalytic efficiency (kcat/Km), consistent with CYP2R1 being the major vitamin D 25-hydroxylase. 20-Hydroxyvitamin D3 [20(OH)D3], the main product of vitamin D3 activation by an alternative pathway catalyzed by CYP11A1, was metabolized by CYP2R1 to 20,25-dihydroxyvitamin D3 [20,25(OH)2D3], with catalytic efficiency similar to that for the 25-hydroxylation of vitamin D3. 20,25(OH)2D3 retained full, or somewhat enhanced activity compared to the parent 20(OH)D3 for the inhibition of the proliferation of melanocytes and dermal fibroblasts, with a potency comparable to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The 20,25(OH)2D3 was also able to act as an inverse agonist on retinoic acid-related orphan receptor α, like its parent 20(OH)D3. Thus, the major findings of this study are that CYP2R1 can metabolize substrates in a membrane environment, the enzyme displays higher catalytic efficiency than CYP27A1 for the 25-hydroxylation of vitamin D, it efficiently hydroxylates 20(OH)D3 at C25 and this product retains the biological activity of the parent compound.


Assuntos
Calcifediol/análogos & derivados , Colestanotriol 26-Mono-Oxigenase/metabolismo , Família 2 do Citocromo P450/metabolismo , Vitaminas/farmacologia , Calcifediol/farmacologia , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colestanotriol 26-Mono-Oxigenase/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Família 2 do Citocromo P450/genética , Escherichia coli/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Hidroxilação , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo
5.
Sci Rep ; 7(1): 10193, 2017 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-28860545

RESUMO

1α,20S-Dihydroxyvitamin D3 [1,20S(OH)2D3], a natural and bioactive vitamin D3 metabolite, was chemically synthesized for the first time. X-ray crystallography analysis of intermediate 15 confirmed its 1α-OH configuration. 1,20S(OH)2D3 interacts with the vitamin D receptor (VDR), with similar potency to its native ligand, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] as illustrated by its ability to stimulate translocation of the VDR to the nucleus, stimulate VDRE-reporter activity, regulate VDR downstream genes (VDR, CYP24A1, TRPV6 and CYP27B1), and inhibit the production of inflammatory markers (IFNγ and IL1ß). However, their co-crystal structures revealed differential molecular interactions of the 20S-OH moiety and the 25-OH moiety to the VDR, which may explain some differences in their biological activities. Furthermore, this study provides a synthetic route for the synthesis of 1,20S(OH)2D3 using the intermediate 1α,3ß-diacetoxypregn-5-en-20-one (3), and provides a molecular and biological basis for the development of 1,20S(OH)2D3 and its analogs as potential therapeutic agents.


Assuntos
Calcifediol/análogos & derivados , Calcifediol/farmacologia , Receptores de Calcitriol/química , Receptores de Calcitriol/metabolismo , Animais , Células CACO-2 , Calcifediol/química , Linhagem Celular , Núcleo Celular/metabolismo , Cristalografia por Raios X , Humanos , Células Jurkat , Modelos Moleculares , Transporte Proteico/efeitos dos fármacos
6.
Artigo em Inglês | MEDLINE | ID: mdl-28622619

RESUMO

25-hydroxyvitamin D3-3-sulfate (25-OHD3-S) and 25-hydroxyvitamin D3-3-glucuronide (25-OHD3-G) are major conjugative metabolites of vitamin D3 found in the systemic circulation and potentially important reservoirs for 25-hydroxyvitamin D3. Simultaneous and accurate quantification of these metabolites could advance assessment of the impact of vitamin D3 on health and disease. In this study, a highly sensitive and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous quantification of 25-OHD3-S and 25-OHD3-G in human serum or plasma. Following protein precipitation, the analytes of interest were partially purified by solid-phase extraction and subjected to derivatization with 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD). Quantification of the analytes was based on multiple reaction monitoring (MRM) operated in the positive ion mode, and deuterated internal standards were used for each conjugative metabolite. Applying this method to the analysis of 25-OHD3-S and 25-OHD3-G concentrations in human serum or plasma samples achieved satisfactory reproducibility, accuracy and sensitivity. We subsequently used this method to simultaneously determine serum concentrations of the two metabolites in archived samples from a rifampin treatment study. Drug treatment had no effect on metabolite concentrations, but significantly increased the 25-OHD3-S/25-OHD3 concentration ratio (p=0.01). The availability of this new method should improve sample throughput and our ability to quantify and monitor circulating 25-OHD3-S and 25-OHD3-G concentrations.


Assuntos
Calcifediol/análogos & derivados , Calcifediol/sangue , Glucuronídeos/sangue , Cromatografia Líquida/métodos , Humanos , Modelos Lineares , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Extração em Fase Sólida , Espectrometria de Massas em Tandem/métodos
7.
J Periodontal Res ; 52(5): 832-841, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28345770

RESUMO

BACKGROUND AND OBJECTIVE: Vitamin D-1,25(OH)2 D3 or 1,25D3-maintains healthy osseous tissue, stimulates the production of the antimicrobial peptide cathelicidin and has anti-inflammatory effects, but it can cause hypercalcemia. Evidence links diminished serum levels of 1,25D3 with increased gingival inflammation. Periodontitis progression is associated with increased local production of inflammatory mediators by immune cells and gingival fibroblasts. These include interleukin (IL)-6, a regulator of osteoclastic bone resorption, and the neutrophil chemoattractant IL-8, both regulated by signaling pathways, including NF-κB and MAPK/AP-1. The objectives were to determine the effects of 1,25D3 or a non-calcemic analog, 20-hydroxyvitamin D3 -20(OH)D3 or 20D3-on IL-1ß-stimulated IL-6 and IL-8 production, and NF-κB and MAPK/AP-1 activation, by human gingival fibroblasts. MATERIAL AND METHODS: Human gingival fibroblasts were incubated ± IL-1ß, with or without exposure to 1,25D3 or 20D3. IL-6 and IL-8 in culture supernatants were measured by enzyme-linked immunosorbent assay. NF-κB (p65) and AP-1 (phospho-cJun) and were measured in nuclear extracts via binding to specific oligonucleotides. Data were analyzed using ANOVA and Scheffe's F procedure for post hoc comparisons. RESULTS: IL-1ß-stimulated IL-6 and IL-8 levels were both significantly inhibited (40%-60%) (P<.045) by 1,25D3, but not 20D3 (0%-15% inhibition, not statistically significant). Both 1,25D3 and 20D3 significantly and similarly inhibited IL-1ß-stimulated nuclear levels of p65 and phospho-cJun (P<.02). CONCLUSION: Reduction of the activation of NF-κB and AP-1 alone is not able to inhibit strongly the IL-1ß stimulated IL-6 and IL-8 gene expression. 1,25D3 but not 20D3 may affect some of the many other factors/processes/pathways that in turn regulate the expression of these genes. However, the results suggest that topical application of ligands of the vitamin D receptor may be useful in the local treatment of periodontitis while reducing adverse systemic effects.


Assuntos
Calcifediol/análogos & derivados , Calcitriol/antagonistas & inibidores , Fibroblastos/efeitos dos fármacos , Gengiva/efeitos dos fármacos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Calcifediol/antagonistas & inibidores , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica , Gengiva/metabolismo , Humanos , Interleucina-6/genética , Interleucina-8/genética , NF-kappa B/efeitos dos fármacos , Periodontite/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição AP-1/efeitos dos fármacos
8.
Oncotarget ; 8(6): 9823-9834, 2017 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-28039464

RESUMO

A novel pathway of vitamin D3 (D3) metabolism, initiated by C20-hydroxylation of D3 by CYP11A1, has been confirmed to operate in vivo. Its major product, 20(OH)D3, exhibits antiproliferative activity in vitro comparable to that of 1,25(OH)2D3, but is noncalcemic in mice and rats. To further characterize the antimelanoma activity of 20(OH)D3, we tested its effect on colony formation of human melanoma cells in monolayer culture and anchorage-independent growth in soft agar. The migratory capabilities of the cells and cell-cell and cell-extracellular matrix interactions were also evaluated using transwell cell migration and spheroid toxicity assays. To assess the antimelanoma activity of 20(OH)D3in vivo, age-matched immunocompromised mice were subcutaneously implanted with luciferase-labelled SKMel-188 cells and were randomly assigned to be treated with either 20(OH)D3 or vehicle (n=10 per group). Tumor size was measured with caliper and live bioimaging methods, and overall health condition expressed as a total body score scale. The following results were observed: (i) 20(OH)D3 inhibited colony formation both in monolayer and soft agar conditions, (ii) 20(OH)D3 inhibited melanoma cells in both transwell migration and spheroid toxicity assays, and (iii) 20(OH)D3 inhibited melanoma tumor growth in immunocompromised mice without visible signs of toxicity. However, although the survival rate was 90% in both groups, the total body score was higher in the treatment group compared to control group (2.8 vs. 2.55). In conclusion, 20(OH)D3, an endogenously produced secosteroid, is an excellent candidate for further preclinical testing as an antimelanoma agent.


Assuntos
Antineoplásicos/farmacologia , Calcifediol/análogos & derivados , Proliferação de Células/efeitos dos fármacos , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Animais , Calcifediol/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Melanoma/patologia , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Cutâneas/patologia , Fatores de Tempo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
9.
J Nutr ; 147(2): 141-151, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27881592

RESUMO

BACKGROUND: The C-3α epimer of 25-hydroxycholecalciferol [3-epi-25(OH)D3] is elevated in infants. OBJECTIVES: We tested whether increasing cholecalciferol intake results in a dose-response in plasma 3-epi-25(OH)D3 We also examined bone and mineral metabolism in response to 3-epi-25(OH)D3 treatment. METHODS: Sprague Dawley rats (4 wk old) were randomly assigned (n = 6/group of each sex) to AIN-93G diets with cholecalciferol at 1 (control), 2, or 4 IU/g diet for objective 1 and to diets with 3-epi-25(OH)D3 at 0.5 or 1 IU/g diet or 25-hydroxycholecalciferol [25(OH)D3] at 0.5 IU/g diet for objective 2 for 8 wk. Measurements at weeks 0, 4, and 8 included body weight and length, plasma vitamin D metabolites, bone biomarkers, and bone mineral density determined by using dual-energy X-ray absorptiometry. Lumbar vertebra 3 (L3) geometry and volumetric bone mineral density (vBMD) were measured using microcomputed tomography. Differences between groups were identified for males and females separately. RESULTS: Weight and food intake were not different between groups. Elevated plasma 3-epi-25(OH)D3 was observed only in females in the 4 IU cholecalciferol/g diet group (mean ± SD: 24.7 ± 17.1 ng/mL), compared with the control group (5.3 ± 1.4 ng/mL; P = 0.001). By week 8, both male and female rats in the 3-epi-25(OH)D3 groups had >87% greater plasma 3-epi-25(OH)D3 concentrations relative to the 25(OH)D3 reference group (P < 0.0001). At week 8 in males only, parathyroid hormone was significantly lower (P = 0.019) in both 3-epi-25(OH)D3 groups than in the 25(OH)D3 group, and L3 total vBMD was higher (P = 0.004) in the 0.5 IU 3-epi-25(OH)D3 group than in the 25(OH)D3 group. CONCLUSIONS: Endogenously generated 3-epi-25(OH)D3 is more prominent in female than in male rats. Exogenous 3-epi-25(OH)D3 was as effective as 25(OH)D3 in supporting bone mineral accretion in both sexes. It thus appears that 3-epi-25(OH)D3 has biological activity and should be further explored.


Assuntos
Densidade Óssea/efeitos dos fármacos , Desenvolvimento Ósseo/efeitos dos fármacos , Calcifediol/análogos & derivados , Calcifediol/farmacologia , Animais , Calcifediol/química , Relação Dose-Resposta a Droga , Feminino , Masculino , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley
10.
J Steroid Biochem Mol Biol ; 159: 131-41, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26970587

RESUMO

20S-Hydroxyvitamin D3 [20(OH)D3] is the biologically active major product of the action of CYP11A1 on vitamin D3 and is present in human plasma. 20(OH)D3 displays similar therapeutic properties to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], but without causing hypercalcaemia and therefore has potential for development as a therapeutic drug. CYP24A1, the kidney mitochondrial P450 involved in inactivation of 1,25(OH)2D3, can hydroxylate 20(OH)D3 at C24 and C25, with the products displaying more potent inhibition of melanoma cell proliferation than 20(OH)D3. CYP3A4 is the major drug-metabolising P450 in liver endoplasmic reticulum and can metabolise other active forms of vitamin D, so we examined its ability to metabolise 20(OH)D3. We found that CYP3A4 metabolises 20(OH)D3 to three major products, 20,24R-dihydroxyvitamin D3 [20,24R(OH)2D3], 20,24S-dihydroxyvitamin D3 [20,24S(OH)2D3] and 20,25-dihydroxyvitamin D3 [20,25(OH)2D3]. 20,24R(OH)2D3 and 20,24S(OH)2D3, but not 20,25(OH)2D3, were further metabolised to trihydroxyvitamin D3 products by CYP3A4 but with low catalytic efficiency. The same three primary products, 20,24R(OH)2D3, 20,24S(OH)2D3 and 20,25(OH)2D3, were observed for the metabolism of 20(OH)D3 by human liver microsomes, in which CYP3A4 is a major CYP isoform present. Addition of CYP3A family-specific inhibitors, troleandomycin and azamulin, almost completely inhibited production of 20,24R(OH)2D3, 20,24S(OH)2D3 and 20,25(OH)2D3 by human liver microsomes, further supporting that CYP3A4 plays the major role in 20(OH)D3 metabolism by microsomes. Since both 20,24R(OH)2D3 and 20,25(OH)2D3 have previously been shown to display enhanced biological activity in inhibiting melanoma cell proliferation, our results show that CYP3A4 further activates, rather than inactivates, 20(OH)D3.


Assuntos
Calcifediol/análogos & derivados , Citocromo P-450 CYP3A/fisiologia , Vias Biossintéticas , Calcifediol/biossíntese , Calcifediol/química , Citocromo P-450 CYP3A/química , Feminino , Humanos , Hidroxilação , Cinética , Masculino , Microssomos Hepáticos/enzimologia
11.
Anticancer Res ; 36(3): 877-86, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26976974

RESUMO

Vitamin D3 (D3) can be metabolized by cytochrome P450scc (CYP11A1) into 20S-hydroxyvitamin D3 (20D3) as a major metabolite. This bioactive metabolite has shown strong antiproliferative, antifibrotic, pro-differentiation and anti-inflammatory effects while being non-toxic (non-calcemic) at high concentrations. Since D3 analogs with two symmetric side chains (Gemini analogs) result in potent activation of the vitamin D receptor (VDR), we hypothesized that the chain length and composition of these types of analogs also containing a 20-hydroxyl group would affect their biological activities. In this study, we designed and synthesized a series of Gemini 20D3 analogs. Biological tests showed that some of these analogs are partial VDR activators and can significantly stimulate the expression of mRNA for VDR and VDR-regulated genes including CYP24A1 and transient receptor potential cation channel V6 (TRPV6). These analogs inhibited the proliferation of melanoma cells with potency comparable to that of 1α,25-dihydroxyvitamin D3. Moreover, these analogs reduced the level of interferon γ and up-regulated the expression of leukocyte associated immunoglobulin-like receptor 1 in splenocytes, indicating that they have potent anti-inflammatory activities. There are no clear correlations between the Gemini chain length and their VDR activation or biological activities, consistent with the high flexibility of the ligand-binding pocket of the VDR.


Assuntos
Anti-Inflamatórios/síntese química , Anti-Inflamatórios/farmacologia , Calcifediol/análogos & derivados , Citostáticos/síntese química , Citostáticos/farmacologia , Animais , Anti-Inflamatórios/química , Calcifediol/síntese química , Calcifediol/química , Calcifediol/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citostáticos/química , Desenho de Fármacos , Humanos , Células Jurkat , Queratinócitos/efeitos dos fármacos , Queratinócitos/imunologia , Camundongos , Receptores de Calcitriol/metabolismo , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologia
12.
Sci Rep ; 5: 14636, 2015 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-26420221

RESUMO

Variations in vitamin D quantification methods are large, and influences of vitamin D analogues and blood collection methods have not been systematically examined. We evaluated the effects of vitamin D analogues 25OHD2 and 3-epi 25OHD3 and blood collection methods on vitamin D measurement, using five immunoassay systems and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Serum samples (332) were selected from routine vitamin D assay requests, including samples with or without 25OHD2 or 3-epi 25OHD3, and analysed using various immunoassay systems. In samples with no 25OHD2 or 3-epi 25OHD3, all immunoassays correlated well with LC-MS/MS. However, the Siemens system produced a large positive mean bias of 12.5 ng/mL and a poor Kappa value when using tubes with clot activator and gel separator. When 25OHD2 or 3-epi 25OHD3 was present, correlations and clinical agreement decreased for all immunoassays. Serum 25OHD in VACUETTE tubes with gel and clot activator, as measured by the Siemens system, produced significantly higher values than did samples collected in VACUETTE tubes with no additives. Bias decreased and clinical agreement improved significantly when using tubes with no additives. In conclusion, most automated immunoassays showed acceptable correlation and agreement with LC-MS/MS; however, 25OHD analogues and blood collection tubes dramatically affected accuracy.


Assuntos
Coleta de Amostras Sanguíneas/instrumentação , Coleta de Amostras Sanguíneas/métodos , Calcifediol/análogos & derivados , Calcifediol/sangue , Imunoensaio/normas , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Automação Laboratorial , Calcifediol/efeitos adversos , Criança , Pré-Escolar , Feminino , Humanos , Imunoensaio/métodos , Lactente , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Adulto Jovem
13.
Steroids ; 104: 153-62, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26433048

RESUMO

A total synthetic strategy of 20S-hydroxyvitamin D3 [20S-(OH)D3] involving modified synthesis of key intermediates 7 and 12, Grignard reaction to stereoselectively generate 20S-OH and Wittig-Horner coupling to establish D3 framework, was completed in 16 steps with an overall yield of 0.4%. The synthetic 20S-(OH)D3 activated vitamin D receptor (VDR) and initiated the expression of downstream genes. In addition, 20S-(OH)D3 showed similar inhibitory potency as calcitriol [1,25(OH)2D3] on proliferation of melanoma cells.


Assuntos
Calcifediol/análogos & derivados , Antineoplásicos/farmacologia , Calcifediol/síntese química , Calcifediol/química , Calcifediol/farmacologia , Calcitriol/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Melanoma/patologia , Estrutura Molecular , Relação Estrutura-Atividade
14.
Endocrinology ; 156(12): 4388-97, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26441239

RESUMO

Vitamin D receptor (VDR)-mediated 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-dependent gene expression is compromised in the VDR null mouse. The biological consequences include: hypocalcemia, hypophosphatemia, elevated parathyroid hormone (PTH) and 1,25(OH)2D3, and consequential skeletal abnormalities. CYP24A1 is a cytochrome P450 enzyme that is involved in the side chain oxidation and destruction of both 1,25(OH)2D3 and 25-hydroxyvitamin D3 (25-OH-D3). In the current studies, we used liquid chromatography-tandem mass spectrometry technology to compare the metabolic profiles of VDR null mice fed either a normal or a calcium and phosphate-enriched rescue diet and to assess the consequence of transgenic expression of either mouse or human VDR genes in the same background. Serum 1,25(OH)2D3 levels in VDR null mice on normal chow were highly elevated (>3000 pg/mL) coincident with undetectable levels of catabolites such as 24,25-(OH)2D3 and 25-OH-D3-26,23-lactone normally observed in wild-type mice. The rescue diet corrected serum Ca(++), PTH, and 1,25(OH)2D3 values and restored basal expression of Cyp24a1 as evidenced by both renal expression of Cyp24a1 and detection of 24,25-(OH)2D3 and the 25-OH-D3-26,23-lactone. Unexpectedly, this diet also resulted in supranormal levels of 3-epi-24,25-(OH)2D3 and 3-epi-25-OH-D3-26,23-lactone. The reappearance of serum 24,25-(OH)2D3 and renal Cyp24a1 expression after rescue suggests that basal levels of Cyp24a1 may be repressed by high PTH. Introduction of transgenes for either mouse or human VDR also normalized vitamin D metabolism in VDR null mice, whereas this metabolic pattern was unaffected by a transgene encoding a ligand binding-deficient mutant (L233S) human VDR. We conclude that liquid chromatography-tandem mass spectrometry-based metabolic profiling is an ideal analytical method to study mouse models with alterations in calcium/phosphate homeostasis.


Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/efeitos dos fármacos , Cálcio na Dieta/farmacologia , Rim/efeitos dos fármacos , Fosfatos/farmacologia , Receptores de Calcitriol/genética , Vitamina D3 24-Hidroxilase/efeitos dos fármacos , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Animais , Calcifediol/análogos & derivados , Calcifediol/metabolismo , Calcitriol/metabolismo , Cromatografia Líquida , Dieta , Regulação da Expressão Gênica , Humanos , Rim/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas em Tandem , Vitamina D/análogos & derivados , Vitamina D/metabolismo , Vitamina D3 24-Hidroxilase/genética , Vitamina D3 24-Hidroxilase/metabolismo
15.
Int J Oncol ; 47(3): 1084-96, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26260259

RESUMO

Colorectal cancer (CRC) is an emerging global problem with the rapid increase in its incidence being associated with an unhealthy lifestyle. Epidemiological studies have shown that decreased levels of vitamin D3 significantly increases the risk of CRC. Furthermore, negative effects of vitamin D3 deficiency can be compensated by appropriate supplementation. Vitamin D3 was shown to inhibit growth and induce differentiation of cancer cells, however, excessive vitamin D3 intake leads to hypercalcemia. Thus, development of efficient vitamin D3 analogues with limited impact on calcium homeostasis is an important scientific and clinically relevant task. The aims of the present study were to compare the antiproliferative potential of classic vitamin D3 metabolites (1α,25(OH)2D3 and 25(OH)D3) with selected low calcemic analogues (calcipotriol and 20(OH)D3) on CRC cell lines and to investigate the expression of vitamin D-related genes in CRC cell lines and clinical samples. Vitamin D3 analogues exerted anti-proliferative effects on all CRC cell lines tested. Calcipotriol proved to be as potent as 1α,25(OH)2D3 and had more efficacy than 20-hydroxyvitamin D3. In addition, the analogs tested effectively inhibited the formation of colonies in Matrigel. The expression of genes involved in 1α,25(OH)2D3 signaling and metabolism varied in cell lines analysed, which explains in part their different sensitivities to the various analogues. In CRC biopsies, there was decreased VDR expression in tumor samples in comparison to the surgical margin and healthy colon samples (p<0.01). The present study indicates that vitamin D3 analogues which have low calcemic activity, such as calcipotriol or 20(OH)D3, are very promising candidates for CRC therapy. Moreover, expression profiling of vitamin D-related genes is likely to be a powerful tool in the planning of anticancer therapy. Decreased levels of VDR and increased CYP24A1 expression in clinical samples underline the importance of deregulation of vitamin D pathways in the development of CRC.


Assuntos
Antineoplásicos/farmacologia , Calcifediol/análogos & derivados , Calcifediol/farmacologia , Calcitriol/análogos & derivados , Neoplasias Colorretais/genética , Calcitriol/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Células HT29 , Humanos , Masculino , Pessoa de Meia-Idade , Receptores de Calcitriol/genética , Transdução de Sinais/efeitos dos fármacos , Vitamina D/análogos & derivados , Vitamina D3 24-Hidroxilase/genética
16.
J Steroid Biochem Mol Biol ; 149: 153-65, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25727742

RESUMO

CYP11A1 hydroxylates vitamin D3 producing 20S-hydroxyvitamin D3 [20(OH)D3] and 20S,23-dihydroxyvitamin D3 [20,23(OH)2D3] as the major and most characterized metabolites. Both display immuno-regulatory and anti-cancer properties while being non-calcemic. A previous study indicated 20(OH)D3 can be metabolized by rat CYP24A1 to products including 20S,24-dihydroxyvitamin D3 [20,24(OH)2D3] and 20S,25-dihydroxyvitamin D3, with both producing greater inhibition of melanoma colony formation than 20(OH)D3. The aim of this study was to characterize the ability of rat and human CYP24A1 to metabolize 20(OH)D3 and 20,23(OH)2D3. Both isoforms metabolized 20(OH)D3 to the same dihydroxyvitamin D species with no secondary metabolites being observed. Hydroxylation at C24 produced both enantiomers of 20,24(OH)2D3. For rat CYP24A1 the preferred initial site of hydroxylation was at C24 whereas the human enzyme preferred C25. 20,23(OH)2D3 was initially metabolized to 20S,23,24-trihydroxyvitamin D3 and 20S,23,25-trihydroxyvitamin D3 by rat and human CYP24A1 as determined by NMR, with both isoforms showing a preference for initial hydroxylation at C25. CYP24A1 was able to further oxidize these metabolites in a series of reactions which included the cleavage of C23-C24 bond, as indicated by high resolution mass spectrometry of the products, analogous to the catabolism of 1,25(OH)2D3 via the C24-oxidation pathway. Similar catalytic efficiencies were observed for the metabolism of 20(OH)D3 and 20,23(OH)2D3 by human CYP24A1 and were lower than for the metabolism of 1,25(OH)2D3. We conclude that rat and human CYP24A1 metabolizes 20(OH)D3 producing only dihydroxyvitamin D3 species as products which retain biological activity, whereas 20,23(OH)2D3 undergoes multiple oxidations which include cleavage of the side chain.


Assuntos
Calcifediol/análogos & derivados , Di-Hidroxicolecalciferóis/metabolismo , Vitamina D3 24-Hidroxilase/metabolismo , Animais , Calcifediol/metabolismo , Humanos , Hidroxilação , Oxirredução , Ratos , Vitamina D/análogos & derivados , Vitamina D/metabolismo
17.
Artigo em Inglês | MEDLINE | ID: mdl-25195024

RESUMO

The quantification of plasma 25-hydroxyvitamin D3 3-sulfate [25(OH)D3S] is expected to be helpful in the assessment of the vitamin D status, especially for infants. In this study, a simple and sensitive method for the quantification of 25(OH)D3S in plasma using liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) has been developed and validated. The plasma was deproteinized with acetonitrile, purified using an Oasis(®) HLB cartridge, and subjected to LC/ESI-MS/MS operating in the negative-ion mode. Quantification was based on the selected reaction monitoring, and deuterated 25(OH)D3S was used as the internal standard. This method enabled the reproducible (intra- and inter-assay relative standard deviations, 7.9% or lower) and accurate (analytical recovery, 95.8-105.3%) quantification of the plasma 25(OH)D3S using a 20-µL sample, and the limit of quantification was 2.5ng/mL. The developed method was applied to the determination of plasma 25(OH)D3S in infants; the result revealed that preterm infants have lower plasma 25(OH)D3S concentrations.


Assuntos
Calcifediol/análogos & derivados , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Adulto , Calcifediol/sangue , Calcifediol/química , Estabilidade de Medicamentos , Idade Gestacional , Humanos , Lactente , Recém-Nascido , Recém-Nascido Prematuro , Modelos Lineares , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
18.
Artigo em Inglês | MEDLINE | ID: mdl-25125396

RESUMO

BACKGROUND: An LC-MS/MS method was developed for simultaneous quantification of 25-hydroxyvitamin D3 (25(OH)D3), 3-epi-25(OH)D3, and 25(OH)D2 in human serum. METHODS: Sample preparation consisted of protein precipitation followed by off-line SPE. Calibration curves for each vitamin D metabolite were constructed in phosphate-buffered saline with 60 g/L albumin including its corresponding stable isotope labelled (SIL) internal standard. A pentafluorophenyl (PFP) analytical column was used to resolve 25(OH)D3 from 25(OH)D2 and 3-epi-25(OH)D3, followed by SRM registration using positive ESI-MS/MS. Accuracy was assessed from measurement of samples with NIST reference method procedure (RMP) assigned values. The PFP LC-MS/MS method was compared to an in-house C18 column LC-MS/MS method, not resolving 25(OH)D3 from 3-epi-25(OH)D3, using adult and newborn samples. RESULTS: Intra-assay and inter-assay coefficients of variation were less than 4% and 7.5%, respectively for all three vitamin D metabolites; lower limits of quantification were 1, 1 and 2 nmol/L and linearity of methods were 1-500, 1-200 and 2-500 nmol/L for 25(OH)D3, 3-epi-25(OH)D3 and 25(OH)D2, respectively. The PFP LC-MS/MS method showed minimal bias to the NIST RMP. Method comparison revealed that in the C18 LC-MS/MS method, the 3-epi-25(OH)D3 concentration is overestimated inadvertently not only from co-elution of both analytes, but also by an additional 30-40% higher ionisation efficiency of 3-epi-25(OH)D3 when compared to 25(OH)D3. CONCLUSION: This accurate LC-MS/MS method allows the simultaneous measurement of 25(OH)D3, 3-epi-25(OH)D3, and 25(OH)D2 in human serum. Due to increased ionisation efficiency, the contribution of the 3-epi-25(OH)D3 metabolite to the total 25(OH)D3 concentration is significantly overestimated in MS methods that do not resolve 3-epi-25(OH)D3 from 25(OH)D3 and may compromise its use in infant samples known to have significant amounts of 3-epi-25(OH)D3.


Assuntos
25-Hidroxivitamina D 2/sangue , 25-Hidroxivitamina D 2/química , Calcifediol/sangue , Calcifediol/química , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , 25-Hidroxivitamina D 2/metabolismo , Adulto , Calcifediol/análogos & derivados , Calcifediol/metabolismo , Humanos , Recém-Nascido , Modelos Lineares , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
19.
J Steroid Biochem Mol Biol ; 144 Pt B: 286-93, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25138634

RESUMO

20-Hydroxyvitamin D3 [20(OH)D3], the major product of CYP11A1 action on vitamin D3, is biologically active and like 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] can inhibit proliferation and promote differentiation of a range of cells, and has anti-inflammatory properties. However, unlike 1,25(OH)2D3, it does not cause toxic hypercalcemia at high doses and is therefore a good candidate for therapeutic use to treat hyperproliferative and autoimmune disorders. In this study we analyzed the ability of mouse liver microsomes to metabolize 20(OH)D3. The two major products were identified from authentic standards as 20,24-dihydroxyvitamin D3 [20,24(OH)2D3] and 20,25-dihydroxyvitamin D3 [20,25(OH)2D3]. The reactions for synthesis of these two products from 20(OH)D3 displayed similar Km values suggesting that they were catalyzed by the same cytochrome P450. Some minor metabolites were produced by reactions with higher Km values for 20(OH)D3. Some metabolites gave mass spectra suggesting that they were the result of hydroxylation followed by dehydrogenation. One product had an increase in the wavelength for maximum absorbance from 263nm seen for 20(OH)D3, to 290nm, suggesting a new double bond was interacting with the vitamin D-triene chromophore. The two major products, 20,24(OH)2D3 and 20,25(OH)2D3 have both previously been shown to have higher potency for inhibition of colony formation by melanoma cells than 20(OH)D3, thus it appears that metabolism of 20(OH)D3 by mouse liver microsomes can generate products with enhanced activity.


Assuntos
Calcifediol/análogos & derivados , Microssomos Hepáticos/metabolismo , Animais , Calcifediol/metabolismo , Feminino , Hidroxilação , Camundongos Endogâmicos C57BL
20.
Am J Clin Nutr ; 100(3): 908-14, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25008852

RESUMO

BACKGROUND: The vitamin D-endocrine system is thought to play a role in physiologic processes that range from mineral metabolism to immune function. Serum 25-hydroxyvitamin D [25(OH)D] is the accepted biomarker for vitamin D status. Skin color is a key determinant of circulating 25(OH)D concentrations, and genes responsible for melanin content have been shown to be under strong evolutionary selection in populations living in temperate zones. Little is known about the effect of latitude on mean concentrations of 25(OH)D in dark-skinned populations. OBJECTIVE: The objective was to describe the distribution of 25(OH)D and its subcomponents in 5 population samples of African origin from the United States, Jamaica, Ghana, South Africa, and the Seychelles. DESIGN: Participants were drawn from the Modeling of the Epidemiologic Transition Study, a cross-sectional observational study in 2500 adults, ages 25-45 y, enrolled between January 2010 and December 2011. Five hundred participants, ∼50% of whom were female, were enrolled in each of 5 study sites: Chicago, IL (latitude: 41°N); Kingston, Jamaica (17°N); Kumasi, Ghana (6°N); Victoria, Seychelles (4°S); and Cape Town, South Africa (34°S). All participants had an ancestry primarily of African origin; participants from the Seychelles trace their history to East Africa. RESULTS: A negative correlation between 25(OH)D and distance from the equator was observed across population samples. The frequency distribution of 25(OH)D in Ghana was almost perfectly normal (Gaussian), with progressively lower means and increasing skewness observed at higher latitudes. CONCLUSIONS: It is widely assumed that lighter skin color in populations outside the tropics resulted from positive selection, driven in part by the relation between sun exposure, skin melanin content, and 25(OH)D production. Our findings show that robust compensatory mechanisms exist that create tolerance for wide variation in circulating concentrations of 25(OH)D across populations, suggesting a more complex evolutionary relation between skin color and the vitamin D pathway.


Assuntos
25-Hidroxivitamina D 2/sangue , Calcifediol/sangue , Modelos Biológicos , Pigmentação da Pele , Pele/efeitos da radiação , Raios Ultravioleta , Deficiência de Vitamina D/epidemiologia , 25-Hidroxivitamina D 2/metabolismo , Adulto , Grupo com Ancestrais do Continente Africano , Biomarcadores/sangue , Calcifediol/análogos & derivados , Calcifediol/metabolismo , Chicago/epidemiologia , Estudos Transversais , Feminino , Gana/epidemiologia , Humanos , Jamaica/epidemiologia , Masculino , Pessoa de Meia-Idade , Risco , Seicheles/epidemiologia , Pele/metabolismo , África do Sul/epidemiologia , Luz Solar , Deficiência de Vitamina D/sangue , Deficiência de Vitamina D/metabolismo
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