Kinetics of liver microsomal cholecalciferol 25-hydroxylase in vitamin D-depleted and -repleated rats.
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ABSTRACT:
SUBMITTER: Delvin EE
PROVIDER: S-EPMC1185715 | biostudies-other | 1978 Jun
REPOSITORIES: biostudies-other
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Project description:The oxygen enzymically inserted as a hydroxy function by rat liver post-mitochondrial fraction into the 25-position of cholecalciferol to giver 25-hydroxycholecaliferol is derived exclusively from molecular O2. Therefore like the other two cholecalciferol hydroxylases, i.e. 25-hydroxycholecalciferol 1alpha-hydroxylase and 25-hydroxycholecalciferol 24-hydroxylase, the cholecalciferol 25-hydroxylase is also a mono-oxygenase ('mixed-function oxidase').
| S-EPMC1186094 | biostudies-other
Project description:The role played by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and/or by calcium on the C-25 hydroxylation of vitamin D3 (D3) was studied in hepatocytes isolated from D-depleted rats which were divided into four treatment groups: Group 1 served as controls, Group 2 received calcium gluconate, Groups 3 and 4 were infused with 1,25(OH)2D3 at 7 and 65 pmol/24 h x 7 days respectively. The treatments normalized serum calcium in all but the controls which remained hypocalcaemic, while serum 1,25(OH)2D3 remained low in Groups 1 and 2 but increased to physiologic and supraphysiologic levels in Groups 3 and 4. The data show that basal D3-25 hydroxylase activities were not significantly affected by any of the treatments. Addition of CaCl2, EGTA, or Quin-2 in vitro revealed that relative to basal values, EGTA strongly inhibited the enzyme activity in all groups (P less than 0.0001), except in G 1; Quin-2 and CaCl2 had no significant effect on the activity of the enzyme in any of the groups. Addition of 1,25(OH)2D3 or A23187 in vitro in the presence of CaCl2 revealed that 1,25(OH)2D3 did not significantly affect enzyme activity, while A23187 was found to stimulate its activity in vitamin D-depleted animals, but most specifically in Group 2 (P less than 0.001); low serum calcium (Group 1) dampened (P less than 0.01), and 1,25(OH)2D3 treatment in vivo totally blunted (P less than 0.001) the response to A23187. The data suggest that 1,25(OH)2D3 supplementation in vivo has per se little or no effect on the basal D3-25 hydroxylase activity. The data show, however, that the magnitude of the response to various challenges in vitro is greatly influenced by the conditioning in vivo of the animals. They also show that A23187 can be a potent stimulator of the enzyme activity, which allowed us to demonstrate a significant reserve for the C-25 hydroxylation of D3 which is well expressed in hepatocytes obtained from D-depleted calcium-supplemented rats.
| S-EPMC1135194 | biostudies-other
Project description:Low plasma level of 25-hydroxyvitamin D (25-OH-D), namely vitamin D deficiency, is associated with obesity and weight loss improves 25-OH-D status. However, the mechanism behind obesity-induced vitamin D deficiency remains unclear. Here, we report that obesity suppresses the expression of cytochrome P450 (CYP) 2R1, the main vitamin D 25-hydroxylase, in both mice and humans. In humans, weight loss induced by gastric bypass surgery increased the expression of CYP2R1 in the s.c. adipose tissue suggesting recovery after the obesity-induced suppression. At the same time, CYP27B1, the vitamin D 1α-hydroxylase, was repressed by the weight loss. In a mouse (C57BL/6N) model of diet-induced obesity, the plasma 25-OH-D was decreased. In accordance, the CYP2R1 expression was strongly repressed in the liver. Moreover, obesity repressed the expression of CYP2R1 in several extrahepatic tissues, the kidney, brown adipose tissue, and testis, but not in the white adipose tissue. Obesity had a similar effect in both male and female mice. In mice, obesity repressed expression of the vitamin D receptor in brown adipose tissue. Obesity also upregulated the expression of the vitamin D receptor and CYP24A1 in the s.c. adipose tissue of a subset of mice; however, no effect was observed in the human s.c. adipose tissue. In summary, we show that obesity affects CYP2R1 expression both in the mouse and human tissues. We suggest that in mouse the CYP2R1 repression in the liver plays an important role in obesity-induced vitamin D deficiency. Currently, it is unclear whether the CYP2R1 downregulation in extrahepatic tissues could contribute to the obesity-induced low plasma 25-OH-D, however, this phenomenon may affect at least the local 25-OH-D concentrations. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
| S-EPMC7657391 | biostudies-literature
Project description:This pilot clinical trial studies cholecalciferol in treating patients with colorectal cancer. The use of cholecalciferol may slow disease progression in patients with colorectal cancer.
| 2110440 | ecrin-mdr-crc
Project description:A cytochrome P-450 which catalyses 25-hydroxylation of vitamin D3 has been purified to apparent homogeneity from pig liver microsomes. The specific content of cytochrome P-450 was 12 nmol.mg of protein-1, and the preparation showed a single band with an apparent M(r) of 50,500 upon SDS/PAGE. A monoclonal antibody raised against the vitamin D3 25-hydroxylase reacted strongly with the purified 25-hydroxylating cytochrome P-450 from pig kidney microsomes [Bergman & Postlind (1990) Biochem. J. 270, 345-350]. The liver enzyme showed structural and functional properties very similar to those of the kidney enzyme. The two enzymes differed with respect to only one of the first 16 N-terminal amino acids. The vitamin D3 25-hydroxylase in pig liver microsomes exhibited a turnover and an apparent Km for 25-hydroxylation of vitamin D3 which were of the same order of magnitude as those of a well-characterized male-specific 25-hydroxylating cytochrome P-450 in rat liver microsomes. The two enzymes differed structurally. The pig liver enzyme was, in contrast to the rat liver enzyme, not sex-specific, and did not catalyse 16 alpha-hydroxylation of testosterone. These properties of the 25-hydroxylase in rat liver microsomes have led to questions on the role of microsomal 25-hydroxylation of vitamin D3. It is concluded that studies on microsomal 25-hydroxylation with the rat may be misleading. The results of the present study show that the pig appears to be a representative species for evaluation of vitamin D3 hydroxylases in other mammals, including man.
| S-EPMC1133068 | biostudies-other
Project description:The conversion of vitamin D into an active ligand for the vitamin D receptor requires 25-hydroxylation in the liver and 1alpha-hydroxylation in the kidney. Mitochondrial and microsomal vitamin D 25-hydroxylase enzymes catalyze the first reaction. The mitochondrial activity is associated with sterol 27-hydroxylase, a cytochrome P450 (CYP27A1); however, the identity of the microsomal enzyme has remained elusive. A cDNA library prepared from hepatic mRNA of sterol 27-hydroxylase-deficient mice was screened with a ligand activation assay to identify an evolutionarily conserved microsomal cytochrome P450 (CYP2R1) with vitamin D 25-hydroxylase activity. Expression of CYP2R1 in cells led to the transcriptional activation of the vitamin D receptor when either vitamin D2 or D3 was added to the medium. Thin layer chromatography and radioimmunoassays indicated that the secosteroid product of CYP2R1 was 25-hydroxyvitamin D3. Co-expression of CYP2R1 with vitamin D 1alpha-hydroxylase (CYP27B1) elicited additive activation of vitamin D3, whereas co-expression with vitamin D 24-hydroxylase (CYP24A1) caused inactivation. CYP2R1 mRNA is abundant in the liver and testis, and present at lower levels in other tissues. The data suggest that CYP2R1 is a strong candidate for the microsomal vitamin D 25-hydroxylase.
| S-EPMC4450819 | biostudies-literature
Project description:Rat liver microsomes (microsomal fraction) were isolated from vitamin A-deficient and -sufficient rats and analysed for membrane lipid characteristics. Membrane fluidity was found to be significantly decreased in microsomes from the vitamin A-deficient rats, but not in liposomes prepared from lipid extracts. Microsomes from vitamin A-deficient animals showed a significant decrease in C18:2, omega 6 and an increase in C22:5, omega 6 fatty acids.
| S-EPMC1148216 | biostudies-other
Project description:Properties of the rat hepatic cholecalciferol 25-hydroxylase have been studied. An assay system has been developed in which 25-hydroxycholecalciferol production is linear for at least 2h in both homogenates and microsomal fraction. Furthermore, the initial reaction velocity is linearly related to the amount of liver tissue or microsomal fraction. This enzyme system also metabolizes an analogue of cholecalciferol, namely dihydrotachysterol 3, into 25-hydroxydihydrotachysterol 3. The 25-hydroxylase is in the microsomal fraction and not in mitochondria. It has a Km of 44 nM for cholecalciferol and 360 nM for dihydrotachysterol 3. Its activity is not altered by dietary concentrations of calcium and phosphorus. Vitamin D-deficient rats have higher activities of the hepatic 25-hydroxylase than those receiving 25 ng of cholecalciferol daily. The 25-hydroxylase is inhibited by metyrapone. An atmosphere of CO/O2 (9:1, v/v) inhibits the reaction by 87%. This inhibition is partially reversed by white light. Additionally, cholecalciferol and 25-hydroxycholecalciferol competitively inhibit aminopyrine demethylase. These results support the idea that the cholecalciferol 25-hydroxylase is a cytochrome P-450-dependent mono-oxygenase.
| S-EPMC1161830 | biostudies-other
Project description:The synthesis of bioactive vitamin D requires hydroxylation at the 1 alpha and 25 positions by cytochrome P450 enzymes in the kidney and liver, respectively. The mitochondrial enzyme CYP27B1 catalyzes 1 alpha-hydroxylation in the kidney but the identity of the hepatic 25-hydroxylase has remained unclear for >30 years. We previously identified the microsomal CYP2R1 protein as a potential candidate for the liver vitamin D 25-hydroxylase based on the enzyme's biochemical properties, conservation, and expression pattern. Here, we report a molecular analysis of a patient with low circulating levels of 25-hydroxyvitamin D and classic symptoms of vitamin D deficiency. This individual was found to be homozygous for a transition mutation in exon 2 of the CYP2R1 gene on chromosome 11p15.2. The inherited mutation caused the substitution of a proline for an evolutionarily conserved leucine at amino acid 99 in the CYP2R1 protein and eliminated vitamin D 25-hydroxylase enzyme activity. These data identify CYP2R1 as a biologically relevant vitamin D 25-hydroxylase and reveal the molecular basis of a human genetic disease, selective 25-hydroxyvitamin D deficiency.
| S-EPMC419671 | biostudies-literature
Project description:Subcellular fractions containing microsomes prepared from rat livers homogenized in the absence of EDTA catalysed the oxidation of cholesterol to 7alpha-hydroxycholesterol, 7-oxocholesterol, 7beta-hydroxycholesterol and 5alpha-cholestane-3beta,5,6beta-triol. These reactions required native protein, molecular oxygen and NADPH. It is suggested that these compounds are formed by a peroxidation analogous to the peroxidation of fatty acids catalysed by liver microsomal preparations. Incubations of [4-(14)C]cholesterol with microsomal preparations from rat liver homogenized in the presence of EDTA gave 7alpha-hydroxy[(14)C]cholesterol as the main product. This reaction required molecular oxygen and NADPH, and was inhibited by CO. The mass of 7alpha-hydroxycholesterol formed during the incubation was measured by a double-isotope-derivative dilution procedure. This procedure was used to assay the activity of cholesterol 7alpha-hydroxylase and to measure low concentrations of endogenous 7alpha-hydroxycholesterol in liver.
| S-EPMC1173563 | biostudies-other