Dataset Information

Differential Metabolic Stability of 4α,25- and 4β,25-Dihydroxyvitamin D₃ and Identification of Their Metabolites.

ABSTRACT: Vitamin D₃ (1) is metabolized by various cytochrome P450 (CYP) enzymes, resulting in the formation of diverse metabolites. Among them, 4α,25-dihydroxyvitamin D₃ (6a) and 4β,25-dihydroxyvitamin D₃ (6b) are both produced from 25-hydroxyvitamin D₃ (2) by CYP3A4. However, 6b is detectable in serum, whereas 6a is not. We hypothesized that the reason for this is a difference in the susceptibility of 6a and 6b to CYP24A1-mediated metabolism. Here, we synthesized 6a and 6b, and confirmed that 6b has greater metabolic stability than 6a. We also identified 4α,24R,25- and 4β,24R,25-trihydroxyvitamin D₃ (16a and 16b) as metabolites of 6a and 6b, respectively, by HPLC comparison with synthesized authentic samples. Docking studies suggest that the β-hydroxy group at C4 contributes to the greater metabolic stability of 6b by blocking a crucial hydrogen-bonding interaction between the C25 hydroxy group and Leu325 of CYP24A1.

SUBMITTER: Mizumoto Y

PROVIDER: S-EPMC10377336 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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Differential Metabolic Stability of 4α,25- and 4β,25-Dihydroxyvitamin D3 and Identification of Their Metabolites.

Mizumoto Yuka Y Sakamoto Ryota R Iijima Kazuto K Nakaya Naoto N Odagi Minami M Tera Masayuki M Hirokawa Takatsugu T Sakaki Toshiyuki T Yasuda Kaori K Nagasawa Kazuo K

Biomolecules 20230624 7

Vitamin D3 (1) is metabolized by various cytochrome P450 (CYP) enzymes, resulting in the formation of diverse metabolites. Among them, 4α,25-dihydroxyvitamin D3 (6a) and 4β,25-dihydroxyvitamin D3 (6b) are both produced from 25-hydroxyvitamin D3 (2) by CYP3A4. However, 6b is detectable in serum, whereas 6a is not. We hypothesized that the reason for this is a difference in the susceptibility of 6a and ...[more]

PMID: 37509072

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Project description:BackgroundGlaucoma is an optic neuropathy characterized by loss of function and death of retinal ganglion cells (RGCs), leading to irreversible vision loss. Neuroinflammation is recognized as one of the causes of glaucoma, and currently no treatment is addressing this mechanism. We aimed to investigate the anti-inflammatory and neuroprotective effects of 1,25(OH)2D3 (1α,25-dihydroxyvitamin D3, calcitriol), in a genetic model of age-related glaucomatous neurodegeneration (DBA/2J mice).MethodsDBA/2J mice were randomized to 1,25(OH)2D3 or vehicle treatment groups. Pattern electroretinogram, flash electroretinogram, and intraocular pressure were recorded weekly. Immunostaining for RBPMS, Iba-1, and GFAP was carried out on retinal flat mounts to assess retinal ganglion cell density and quantify microglial and astrocyte activation, respectively. Molecular biology analyses were carried out to evaluate retinal expression of pro-inflammatory cytokines, pNFκB-p65, and neuroprotective factors. Investigators that analysed the data were blind to experimental groups, which were unveiled after graph design and statistical analysis, that were carried out with GraphPad Prism. Several statistical tests and approaches were used: the generalized estimated equations (GEE) analysis, t-test, and one-way ANOVA.ResultsDBA/2J mice treated with 1,25(OH)2D3 for 5 weeks showed improved PERG and FERG amplitudes and reduced RGCs death, compared to vehicle-treated age-matched controls. 1,25(OH)2D3 treatment decreased microglial and astrocyte activation, as well as expression of inflammatory cytokines and pNF-κB-p65 (p < 0.05). Moreover, 1,25(OH)2D3-treated DBA/2J mice displayed increased mRNA levels of neuroprotective factors (p < 0.05), such as BDNF.Conclusions1,25(OH)2D3 protected RGCs preserving retinal function, reducing inflammatory cytokines, and increasing expression of neuroprotective factors. Therefore, 1,25(OH)2D3 could attenuate the retinal damage in glaucomatous patients and warrants further clinical evaluation for the treatment of optic neuropathies.

Project description:We previously demonstrated that non-small cell lung cancer (NSCLC) cells and primary human lung tumors aberrantly express the vitamin D3-catabolizing enzyme, CYP24, and that CYP24 restricts transcriptional regulation and growth control by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) in NSCLC cells. To ascertain the basis for CYP24 dysregulation, we assembled a panel of cell lines that represent distinct molecular classes of lung cancer: cell lines were selected which harbored mutually exclusive mutations in either the K-ras or the Epidermal Growth Factor Receptor (EGFR) genes. We observed that K-ras mutant lines displayed a basal vitamin D receptor (VDR)(low)CYP24(high) phenotype, whereas EGFR mutant lines had a VDR(high)CYP24(low) phenotype. A mutation-associated difference in CYP24 expression was also observed in clinical specimens. Specifically, K-ras mutation was associated with a median 4.2-fold increase in CYP24 mRNA expression (p=4.8×10(-7)) compared to EGFR mutation in a series of 147 primary lung adenocarcinoma cases. Because of their differential basal expression of VDR and CYP24, we hypothesized that NSCLC cells with an EGFR mutation would be more responsive to 1,25(OH)2D3 treatment than those with a K-ras mutation. To test this, we measured the ability of 1,25(OH)2D3 to increase reporter gene activity, induce transcription of endogenous target genes, and suppress colony formation. In each assay, the extent of 1,25(OH)2D3 response was greater in EGFR mutation-positive HCC827 and H1975 cells than in K-ras mutation-positive A549 and 128.88T cells. We subsequently examined the effect of combining 1,25(OH)2D3 with erlotinib, which is used clinically in the treatment of EGFR mutation-positive NSCLC. 1,25(OH)2D3/erlotinib combination resulted in significantly greater growth inhibition than either single agent in both the erlotinib-sensitive HCC827 cell line and the erlotinib-resistant H1975 cell line. These data are the first to suggest that EGFR mutations may identify a lung cancer subset which remains responsive to and is likely to benefit from 1,25(OH)2D3 administration. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

Dataset Information

Differential Metabolic Stability of 4α,25- and 4β,25-Dihydroxyvitamin D₃ and Identification of Their Metabolites.

Publications

Differential Metabolic Stability of 4α,25- and 4β,25-Dihydroxyvitamin D<sub>3</sub> and Identification of Their Metabolites.

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