| 1. |
- Araya, Zufan, et al.
(author)
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Metabolism of 25-hydroxyvitamin D3 by microsomal and mitochondrial vitamin D3 25-hydroxylases (CYP2D25 and CYP27A1) : a novel reaction by CYP27A1
- 2003
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In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids. - 1388-1981 .- 1879-2618. ; 1632:1-21-3, s. 40-47
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Journal article (peer-reviewed)abstract
- The metabolism of 25-hydroxyvitamin D(3) was studied with a crude mitochondrial cytochrome P450 extract from pig kidney and with recombinant human CYP27A1 (mitochondrial vitamin D(3) 25-hydroxylase) and porcine CYP2D25 (microsomal vitamin D(3) 25-hydroxylase). The kidney mitochondrial cytochrome P450 catalyzed the formation of 1alpha,25-dihydroxyvitamin D(3), 24,25-dihydroxyvitamin D(3) and 25,27-dihydroxyvitamin D(3). An additional metabolite that was separated from the other hydroxylated products on HPLC was also formed. The formation of this 25-hydroxyvitamin D(3) metabolite was dependent on NADPH and the mitochondrial electron transferring protein components. A monoclonal antibody directed against purified pig liver CYP27A1 immunoprecipitated the 1alpha- and 27-hydroxylase activities towards 25-hydroxyvitamin D(3) as well as the formation of the unknown metabolite. These results together with substrate inhibition experiments indicate that CYP27A1 is responsible for the formation of the unknown 25-hydroxyvitamin D(3) metabolite in kidney. Recombinant human CYP27A1 was found to convert 25-hydroxyvitamin D(3) into 1alpha,25-dihydroxyvitamin D(3), 25,27-dihydroxyvitamin D(3) and a major metabolite with the same retention time on HPLC as that formed by kidney mitochondrial cytochrome P450. Gas chromatography-mass spectrometry (GC-MS) analysis of the unknown enzymatic product revealed it to be a triol different from other known hydroxylated 25-hydroxyvitamin D(3) metabolites such as 1alpha,25-, 23,25-, 24,25-, 25,26- or 25,27-dihydroxyvitamin D(3). The product had the mass spectrometic properties expected for 4beta,25-dihydroxyvitamin D(3). Recombinant porcine CYP2D25 converted 25-hydroxyvitamin D(3) into 1alpha,25-dihydroxyvitamin D(3) and 25,26-dihydroxyvitamin D(3). It can be concluded that both CYP27A1 and CYP2D25 are able to carry out multiple hydroxylations of 25-hydroxyvitamin D(3).
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| 2. |
- Hosseinpour, Fardin, et al.
(author)
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25-Hydroxylation of vitamin D3 in primary cultures of pig hepatocytes:evidence for a role of both CYP2D25 and CYP27A1
- 2003
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In: Biochemical and Biophysical Research Communications - BBRC. - 0006-291X .- 1090-2104. ; 303:3, s. 877-883
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Journal article (peer-reviewed)abstract
- There has been some controversy over whether the 25-hydroxylation of vitamin D(3) is carried out by one enzyme or two and whether this cytochrome P450 enzyme is found in the mitochondrial or microsomal fractions of liver. The pig is currently the only species in which both the microsomal 25-hydroxylase (CYP2D25) and the mitochondrial 25-hydroxylase (CYP27A1) have been cloned and characterized. In this paper, the roles of the two enzymes in 25-hydroxylation of vitamin D(3) are examined in primary cultures of hepatocytes. Inhibition experiments indicated that tolterodine and 7 alpha-hydroxy-4-cholesten-3-one were selective inhibitors of the CYP2D25- and CYP27A-mediated 25-hydroxylation of vitamin D(3), respectively. Addition of each inhibitor to primary hepatocytes decreased the total 25-hydroxylation of vitamin D(3) to about the same extent. No inhibition of other hydroxylase activities tested was found. Phorbol 12-myristate 13-acetate down-regulated the expression of both CYP2D25 and CYP27A1 as well as the 25-hydroxylase activity of the hepatocytes. The results implicate that both CYP2D25 and CYP27A1 contribute to the 25-hydroxylation in hepatocytes and are important in the bioactivation of vitamin D(3).
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| 3. |
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| 4. |
- Hosseinpour, Fardin, 1965-
(author)
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Cytochrome P450 Enzymes in the Metabolism of Vitamin D3
- 2002
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Doctoral thesis (other academic/artistic)abstract
- A cytochrome P450 enzyme in pig kidney that catalyzes the hydroxylations of vitamin D3 and C27-sterols was cloned. DNA sequence analysis of the cDNA revealed that the enzyme belongs to the CYP27 family. The recombinant kidney CYP27A enzyme catalyzed the 25-hydroxylation of vitamin D3 and the 27-oxygenation of C27-sterols. It was shown that human embryonic kidney cells express CYP27A mRNA and are able to catalyze the same reactions. Microsomal vitamin D3 25-hydroxylase (CYP2D25), purified from pig liver, converted vitamin D3 into 25- hydroxyvitamin D3 in substrate concentrations which are within the physiological range. The enzyme also converted tolterodine, a substrate for CYP2D6, into its 5-hydroxymethyl metabolite. RT-PCR experiments revealed that CYP2D25 mRNA is expressed not only in liver and kidney but also in other organs. Experiments with human liver microsomes and recombinant human CYP2D6 indicate that the microsomal 25-hydroxylation of vitamin D3 in human liver is catalyzed by an enzyme different from CYP2D6. Five residues in SRS-3 of CYP2D25 were simultaneously mutated to the equivalent residues in CYP2D6, an enzyme not active in 25-hydroxylation. Both wild-type and mutated CYP2D25 were expressed in the Saccharomyces cerevisiae W(R) strain. The 25-hydroxylase activity of recombinant mutant CYP2D25 was completely lost whereas the activity toward tolterodine remained unaffected. These results indicate that residues in SRS-3 of CYP2D25 are important determinants for its function in vitamin D3 metabolism. A cDNA homologous with the hepatic CYP2D25 was cloned from pig kidney. The enzyme purified from pig kidney and the recombinant enzyme expressed in COS cells catalyzed 25-hydroxylation of vitamin D3 and, in addition, lα-hydroxylation of 25-hydroxyvitamin D3. Immunohistochemistry experiments indicate that CYP2D25 is expressed almost exclusively in the cells of cortical proximal tubules. The expression of CYP2D25 in kidney, but not in liver, was much higher in the adult pig than in the newborn. The results imply that CYP2D25 has a biological role in kidney. Results from experiments with inhibitors in primary cultures of porcine hepatocytes suggest that both CYP2D25 and CYP27A1 contribute to the total 25-hydroxylation in hepatocytes and are equally important in the bioactivation of vitamin D3. Phenobarbital treatment increased the CYP2D25 mRNA levels but did not affect the CYP27A1 mRNA levels. The rate of 25-hydroxylation by phenobarbital-treated hepatocytes was markedly reduced. These results show that primary cultures of porcine hepatocytes are suitable as a model to study the metabolism of vitamin D3 and the regulation of the CYP enzymes involved in the 25-hydroxylation o vitamin D3.
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| 5. |
- Hosseinpour, Fardin, et al.
(author)
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Phenobarbital suppresses vitamin D3 25-hydroxylase expression : A potential new mechanism for drug-induced osteomalacia
- 2007
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In: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 357:3, s. 603-607
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Journal article (peer-reviewed)abstract
- Prolonged therapy with phenobarbital may cause vitamin D deficiency or osteomalacia. In the current study, we propose a novel mechanism for drug-induced osteomalacia involving impaired bioactivation of vitamin D3 due to decreased 25-hydroxylation of vitamin D3 in liver. The present data, using the pig as model, demonstrate direct effects by phenobarbital on the expression of CYP27A1 and CYP2D25, two important 25-hydroxylases. Treatment by phenobarbital markedly reduced the rate of 25-hydroxylation by primary hepatocytes and suppressed the cellular CYP27A1 mRNA levels. The rate of 25-hydroxylation by two different purified 25-hydroxylases, microsomal CYP2D25, and mitochondrial CYP27A1, respectively, was dose-dependently inhibited by phenobarbital. Reporter assay experiments in liver-derived HepG2 cells revealed a marked PXR-mediated transcriptional downregulation of the CYP2D25 promoter. In addition, the data indicate that phenobarbital might affect the mRNA stability of CYP2D25. Taken together, the data suggest that vitamin D3 25-hydroxylation may be suppressed by phenobarbital. A downregulation of 25-hydroxylation by phenobarbital may explain, at least in part, the increased risk of osteomalacia, bone loss, and fractures in long-term phenobarbital therapy.
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| 6. |
- Hosseinpour, Fardin, et al.
(author)
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Porcine microsomal vitamin D-3 25-hydroxylase (CYP2D25) : Catalytic properties, tissue distribution, and comparison with human CYP2D6
- 2000
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 275:44, s. 34650-34655
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Journal article (peer-reviewed)abstract
- The metabolic activation of the prohormone vitamin D(3) requires a 25-hydroxylation that has been reported to be catalyzed by both mitochondrial CYP27A and a microsomal vitamin D(3) 25-hydroxylase in the liver. CYP27A has been extensively studied, but its role as a physiologically important vitamin D(3) 25-hydroxylase has been questioned. The present paper reports that the microsomal vitamin D(3) 25-hydroxylase, purified from pig liver, converted vitamin D(3) into 25-hydroxyvitamin D(3) in substrate concentrations which are within the physiological range (apparent K(m) = 0.1 microm). The enzyme 25-hydroxylated vitamin D(3), 1 alpha-hydroxyvitamin D(3) and vitamin D(2) and also converted tolterodine, a substrate for human CYP2D6, into its 5-hydroxymethyl metabolite. Tolterodine inhibited the microsomal 25-hydroxylation, whereas quinidine, an inhibitor of CYP2D6, did not markedly inhibit the reaction. The primary structure of the microsomal vitamin D(3) 25-hydroxylase, designated CYP2D25, shows 77% identity with that of human CYP2D6. Northern blot and reverse transcription-polymerase chain reaction experiments revealed that CYP2D25 mRNA is expressed in higher levels in liver than in kidney and in small amounts in adrenals, brain, heart, intestine, lung, muscle, spleen, and thymus. Experiments with human liver microsomes and recombinantly expressed CYP2D6 strongly indicate that the microsomal 25-hydroxylation of vitamin D(3) in human liver is catalyzed by an enzyme different from CYP2D6.
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