Metabolism of 25-hydroxyvitamin D3 in rats: low-calcium diet vs. calcitriol infusion

1992 ◽  
Vol 262 (3) ◽  
pp. E359-E367 ◽  
Author(s):  
M. J. Bolt ◽  
W. E. Jensen ◽  
M. D. Sitrin
Keyword(s):  
Side Chain ◽  
Metabolic Clearance ◽  
Chain Oxidation ◽  
Hydroxyvitamin D ◽  
Dihydroxyvitamin D ◽  
Low Calcium Diet ◽  
Subsequent Degradation ◽  
Dramatic Rise ◽  
25 Hydroxyvitamin D

It has been proposed that the decreased serum level of 25-hydroxyvitamin D [25(OH)D] observed with dietary Ca restriction is mediated by an increase in circulating 1,25-dihydroxyvitamin D [1,25(OH)2D]. We compared the effects of endogenous and exogenous elevations in serum 1,25(OH)2D on the production rate (PR), metabolic clearance rate (MCR), and excretory pathways of [3H]25(OH)D3 in rats, with the use of steady-state techniques. Low-Ca diet and 1,25(OH)2D3 infusion caused comparable reductions in serum 25(OH)D and elevations in 1,25(OH)2D. Low-Ca diet lowered serum 25(OH)D by increasing MCR from 21.8 +/- 3.2 to 29.1 +/- 5.4 (SD) microliters.min-1.kg-1 (P less than or equal to 0.005) and decreasing PR from 944 +/- 161 to 663 +/- 163 pg.Ain-1.kg-1 (P less than or equal to 0.001). In contrast, 1,25(OH)2D3 infusion produced a dramatic rise in the MCR of 25(OH)D from 23.4 +/- 4.5 to 62.8 +/- 13.7 microliters.min-1.kg-1 (P less than or equal to 0.001) and also increased the PR from 943 +/- 165 to 1,500 +/- 337 pg.min-1.kg-1 (P less than or equal to 0.001). With 1,25(OH)2D3 infusion, urinary excretion of metabolites of [3H]25(OH)D3 rose rapidly, and kidney homogenates from these rats demonstrated vigorous side-chain oxidation of [3H]25(OH)D3. With low-Ca diet, urinary tritium excretion increased more gradually, and no direct side-chain oxidation of [3H]25(OH)D3 occurred in vitro. The increased MCR of 25(OH)D3 with low-Ca diet could be accounted for by enhanced synthesis of 1,25(OH)2D3 and subsequent degradation in target tissues.

Side-chain oxidation of vitamin D3 in mouse kidney mitochondria: effect of the Hyp mutation and 1,25-dihydroxyvitamin D3 treatment

1987 ◽  
Vol 65 (10) ◽  
pp. 853-859 ◽  
Author(s):  
Glenville Jones ◽  
Agatha Yip ◽  
Harriet S. Tenenhouse
Keyword(s):  
Enzyme Activity ◽  
Mouse Kidney ◽  
Side Chain ◽  
Mitochondrial Enzyme ◽  
Renal Metabolism ◽  
Chain Oxidation ◽  
Hydroxyvitamin D ◽  
Dihydroxyvitamin D ◽  
1,25 Dihydroxyvitamin D ◽  
25 Hydroxyvitamin D

Side-chain oxidation of vitamin D is an important degradative pathway. In the present study we compared the enzymes involved in side-chain oxidation in normal and Hyp mouse kidney. Homogenates of normal mouse kidney catalyze the conversion of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3, 24-oxo-25-hydroxyvitamin D3, and 24-oxo-23,25-dihydroxyvitamin D3. After subcellular fractionation, total side-chain oxidative activity, estimated by the sum of the three products synthesized per milligram protein under initial rate conditions, coincided with the mitochondrial enzyme marker succinate–cytochrome-c reductase. Treatment of normal mice with 1,25-dihydroxyvitamin D3 (1.5 ng/g) resulted in an eightfold increase in mitochondrial enzyme activity, with no change in apparent Km but a significant rise in Vmax. With 24,25-dihydroxyvitamin D3 as the substrate, normal renal mitochondria produced 24-oxo-25-hydroxyvitamin D3 and 24-oxo-23,25-dihydroxyvitamin D3, and the synthesis of these metabolites could be increased sixfold by pretreatment with 1,25-dihydroxyvitamin D3. In the Hyp mouse, the side-chain oxidation pathway showed similar subcellular distribution of enzyme activity. However, product formation from 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 was twofold greater in mutant than in normal mitochondria. Furthermore, 1,25-dihydroxyvitamin D3 pretreatment of Hyp mice resulted in a 3.4-fold increase over basal metabolism of both 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3. These results demonstrate that (i) kidneys from normal and Hyp mice possess basal and 1,25-dihydroxyvitamin D3 inducible enzyme system(s) in the mitochondrial fraction, which catalyze the side-chain oxidation of 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3, and (ii) the Hyp mutation appears to perturb the renal metabolism of both substrates only in the basal state.

Role of 25-hydroxyvitamin D3 dose in determining rat 1,25-dihydroxyvitamin D3 production

1990 ◽  
Vol 258 (5) ◽  
pp. E780-E789 ◽  
Author(s):  
R. Vieth ◽  
K. McCarten ◽  
K. H. Norwich
Keyword(s):  
Specific Activity ◽  
Metabolic Clearance ◽  
Dihydroxyvitamin D3 ◽  
Hydroxyvitamin D ◽  
Dihydroxyvitamin D ◽  
25 Hydroxyvitamin D ◽  
Dose Dependent

To understand the relationships among 1) the dose of 25-hydroxyvitamin D [25(OH)D] in vivo, 2) the activity of 1-hydroxylase in renal mitochondria, and 3) the production of 1,25-dihydroxyvitamin D [1,25(OH)2D] in vivo, we gave rats different chronic or acute doses of 25-hydroxyvitamin D3 [25(OH)D3]. We followed the metabolism of intracardially administered [25-hydroxy-26,27-methyl-3H]cholecalciferol [25(OH)[3H]D3] for 24 h before killing by measuring extracts of serum by chromatography. Specific activity of 1-hydroxylase in kidney was measured at death. In rats given 0-2,000 pmol 25(OH)D3 chronically by mouth, there was a dose-dependent decline in the percent of serum radioactivity made up of 1,25-dihydroxy-[26,27-methyl-3H]cholecalciferol [1,25(OH)2[3H]D3] as well as a decline in mitochondrial 1-hydroxylase, and these correlated significantly (r = 0.83, P less than 0.001). Serum %1,25(OH)2[3H]D3 in this experiment ranged from 0.8 to 42%. A small part of this range could be accounted for by a faster metabolic clearance rate (MCR) of 1,25(OH)2D3 from rats supplemented with 25(OH)D3 (MCR, 2.12 +/- 0.10 ml/min) compared with rats restricted in vitamin D (MCR, 0.94 +/- 0.06 ml/min, P less than 0.001). The activity of 1-hydroxylase was by far the major factor determining serum %1,25(OH)2[3H]D3. When different acute doses of 25(OH)D3 were given to rats with identical specific activities of 1-hydroxylase, the resulting 1,25(OH)2D3 concentrations in serum correlated with the 25(OH)D3 dose (r = 0.99, P less than 0.001). We conclude that the behavior of 1-hydroxylase in vivo is analogous to the classic behavior in vitro of an enzyme functioning below its Michaelis constant (Km). The amount of 1-hydroxylase present in renal mitochondria determines the fraction (not simply the quantity) of 25(OH)D metabolized to 1,25(OH)2D3 in vivo.

Metabolism of 25-hydroxyvitamin D in copper-laden rat: a model of Wilson's disease

1988 ◽  
Vol 254 (2) ◽  
pp. E150-E154
Author(s):  
T. O. Carpenter ◽  
M. L. Pendrak ◽  
C. S. Anast
Keyword(s):  
Vitamin D ◽  
Wilson’S Disease ◽  
Wilson's Disease ◽  
Vitamin D Metabolism ◽  
Hydroxyvitamin D ◽  
Dihydroxyvitamin D ◽  
Hepatic Copper ◽  
25 Hydroxyvitamin D ◽  
Potential Factor

Wilson's disease results in excess tissue accumulation of copper and is often complicated by skeletal and mineral abnormalities. We investigated vitamin D metabolism in rats fed a copper-laden diet rendering hepatic copper content comparable with that found in Wilson's disease. Injection of 25-hydroxyvitamin D3 [25(OH)D3] resulted in reduced 1,25-dihydroxyvitamin D [1,25(OH)2D] levels in copper-intoxicated rats. In vitro 25(OH)D-1 alpha-hydroxylase activity was impaired in renal mitochondria from copper-intoxicated animals. Activity was also inhibited in mitochondria from controls when copper was added to incubation media. Impaired conversion of 25(OH)D to 1,25(OH)2D occurs in copper intoxication and suggests that altered vitamin D metabolism is a potential factor in the development of bone and mineral abnormalities in Wilson's disease.

Vitamin D kinetics in vivo: effect of 1,25-dihydroxyvitamin D administration

1989 ◽  
Vol 256 (5) ◽  
pp. E686-E691
Author(s):  
B. P. Halloran ◽  
M. E. Castro
Keyword(s):  
Serum Concentration ◽  
Chronic Administration ◽  
Metabolic Clearance ◽  
Metabolic Clearance Rate ◽  
Hydroxyvitamin D ◽  
Dihydroxyvitamin D ◽  
1,25 Dihydroxyvitamin D ◽  
25 Hydroxyvitamin D ◽  
Kinetics In Vivo

Administration of 1,25-dihydroxyvitamin D [1,25(OH)2D] can increase the metabolic clearance rate (MCR) of 25-hydroxyvitamin D [25(OH)D]. To determine whether administration of 1,25(OH)2D can also influence the metabolic clearance rates (MCR) of 1,25(OH)2D and 24,25-dihydroxyvitamin D 24,25(OH)2D, we measured metabolic clearance of 1,25(OH)2D, 24,25(OH)2D, and 25(OH)D in rats in which the serum concentration of 1,25(OH)2D was increased by continuous infusion. Infusion of 1,25(OH)2D (12 days at 75 pmol/day) increased serum 1,25(OH)2D from 128 +/- 11 to 244 +/- 14 pg/ml (P less than 0.005) and increased MCR from 169 +/- 13 to 210 +/- 9 microliters.min-1.kg-1 or 24% (P less than 0.025). Increasing serum 1,25(OH)2D to 330-360 pg/ml increased MCR 72%. Infusion of 1,25(OH)2D decreased serum 24,25(OH)2D from 3.5 +/- 0.5 to 2.4 +/- 0.3 ng/ml (P less than 0.05), increased MCR from 25 +/- 2 to 48 +/- 6 microliters.min-1.kg-1 (P less than 0.0025), and increased the production rate (PR) from 70 +/- 11 to 124 +/- 26 pg.min-1.kg-1 (P less than 0.05). Infusion of 1,25(OH)2D decreased serum 25(OH)D from 13.0 +/- 0.5 to 8.0 +/- 0.5 ng/ml (P less than 0.005) and increased MCR from 45 +/- 1 to 75 +/- 7 microliters.min-1.kg-1 (P less than 0.001) but had no effect on PR. The data indicate that increasing serum 1,25(OH)2D by chronic administration can increase the MCR of 1,25(OH)2D and suggest that 1,25(OH)2D can feedback regulate its serum concentration by regulating its MCR. The data also suggest that 1,25(OH)2D administration can increase the MCRs of 24,25(OH)2D and 25(OH)D.

scholarly journals Cloning of Porcine 25-Hydroxyvitamin D3 1α-Hydroxylase and Its Regulation by cAMP in LLC-PK1 Cells

1999 ◽  
Vol 10 (5) ◽  
pp. 963-970
Author(s):  
TADASHI YOSHIDA ◽  
NORIKO YOSHIDA ◽  
AKIRA NAKAMURA ◽  
TOSHIAKI MONKAWA ◽  
MATSUHIKO HAYASHI ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Cytochrome P450 Enzyme ◽  
Pcr Cloning ◽  
Humoral Factors ◽  
Hydroxyvitamin D ◽  
Dihydroxyvitamin D ◽  
Reverse Transcription Pcr ◽  
25 Hydroxyvitamin D ◽  
Renal Proximal Tubular Cells

Abstract. The 25-hydroxyvitamin D3 1α-hydroxylase, also referred to as CYP27B1, is a mitochondrial cytochrome P450 enzyme that catalyzes the biosynthesis of 1α, 25-dihydroxyvitamin D3 (1α,25(OH)2D3) from 25-hydroxyvitamin D3 in renal proximal tubular cells. Recently, human, mouse, and rat CYP27B1 cDNA have been cloned, however the gene regulation has not been fully elucidated. In the present study, porcine CYP27B1 cDNA was cloned, and the effects of cAMP and vitamin D3 on the regulation of CYP27B1 mRNA expression in LLC-PK1 cells were examined. PCR cloning revealed that porcine CYP27B1 cDNA consisted of 2316 bp, encoding a protein of 504 amino acids. The deduced amino acid sequence showed over 80% identity to the human, mouse, and rat enzyme. LLC-PK1 cells were incubated with humoral factors, and expression of CYP27B1 mRNA was measured by a quantitative reverse transcription-PCR. At the completion of 3-, 6-, 12-, and 24-h incubations, 500 μmol/L 8-bromo-cAMP had significantly increased CYP27B1 mRNA expression (260 to 340%). The adenylate cyclase activator forskolin at 50 μmol/L also had a stimulatory effect at 6 h (190%). Moreover, the protein kinase A inhibitor H-89 reduced the cAMP effect. On the other hand, 1α,25(OH)2D3 had no effect on CYP27B1 mRNA expression at 10 and 100 nmol/L, whereas expression of 25-hydroxyvitamin D3 24-hydroxylase (CYP24) mRNA was markedly increased by 1α,25(OH)2D3. These findings suggest that LLC-PK1 cells express CYP27B1 mRNA, and that cAMP is an upregulating factor of the CYP27B1 gene in vitro.

Metabolism of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3 and its sensitivity to ketoconazole in 1α,25-dihydroxyvitamin D3-differentiated HL60 cells

1989 ◽  
Vol 3 (3) ◽  
pp. 199-205 ◽  
Author(s):  
M. E. Hayes ◽  
D. Bayley ◽  
E. B. Mawer
Keyword(s):  
High Performance ◽  
Cell Number ◽  
Dependent Manner ◽  
Macrophage Phenotype ◽  
Dihydroxyvitamin D3 ◽  
Hydroxyvitamin D ◽  
Dihydroxyvitamin D ◽  
25 Hydroxyvitamin D ◽  
Dose Dependent

ABSTRACT Regulation of the metabolism of [3H]25-hydroxyvitamin D3 ([3H]25-(OH)D3) in vitro to material with the characteristics of [3H]24,25-dihydroxyvitamin D3 ([3H]24,25-(OH)2D3) has been studied in the human promyelocytic cell line HL60. Synthesis of 24,25-(OH)2D3 was induced in a dose-dependent manner in cells pretreated with 0·1–100 nm 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2D3) for 4 days. This treatment also inhibited cell proliferation and stimulated differentiation to a macrophage phenotype that was characterized by staining for non-specific esterase (NSE) activity. The ability to synthesize [3H]24,25-(OH)2D3 from [3H]25-(OH)D3 and the expression of NSE activity both responded to changes in concentration of 1α,25-(OH)2D3 in the culture medium in a parallel manner. Synthesis of [3H]24,25-(OH)2D3 was linear when the incubation time was between 1 and 8 h and the cell number between 1 and 12×106 cells/incubation. The optimum substrate concentration for its synthesis was 125 nm, giving an apparent Michaelis constant of 360 nm. The identity of the [3H]24,25-(OH)2D3 synthesized by these cells was confirmed by co-chromatography with authentic 24,25-(OH)2D3 on normal-phase and reverse-phase high-performance liquid chromatography systems and by its reaction to sodium-m-periodate. Cells that had been exposed to 100 nm 1α,25-(OH)2D3 for 4 days synthesized 2·17±0·07 (s.e.m.) pmol 24,25-(OH)2D3/106 cells per h. This synthesis was inhibited in a dose-dependent manner over a concentration range of 0·01–1 μm by the drug ketoconazole, an antimycotic imidazole which is a known inhibitor of certain cytochrome P-450 enzyme systems, suggesting that the HL60 25-(OH)D3-24-hydroxylase is also a P-450-dependent enzyme system.

scholarly journals Age-related alteration of vitamin D metabolism in response to low-phosphate diet in rats

2005 ◽  
Vol 93 (3) ◽  
pp. 299-307 ◽  
Author(s):  
Tsui-Shan Chau ◽  
Wan-Ping Lai ◽  
Pik-Yuen Cheung ◽  
Murray J. Favus ◽  
Man-Sau Wong
Keyword(s):  
Vitamin D ◽  
Mrna Expression ◽  
Age Groups ◽  
Metabolic Clearance ◽  
Adult Rats ◽  
Vitamin D Metabolism ◽  
Hydroxyvitamin D ◽  
Dihydroxyvitamin D ◽  
Age Related ◽  
25 Hydroxyvitamin D

The responses of renal vitamin D metabolism to its major stimuli alter with age. Previous studies showed that the increase in circulating 1,25-dihydroxyvitamin D (1,25(OH)2D3) as well as renal 25-hydroxyvitamin D3 1-α hydroxylase (1-OHase) activity in response to dietary Ca or P restriction reduced with age in rats. We hypothesized that the mechanism involved in increasing circulating 1,25(OH)2D3 in response to mineral deficiency alters with age. In the present study, we tested the hypothesis by studying the expression of genes involved in renal vitamin D metabolism (renal 1-OHase, 25-hydroxyvitamin D 24-hydroxylase (24-OHase) and vitamin D receptor (VDR)) in young (1-month-old) and adult (6-month-old) rats in response to low-phosphate diet (LPD). As expected, serum 1,25(OH)2D3 increased in both young and adult rats upon LPD treatment and the increase was much higher in younger rats. In young rats, LPD treatment decreased renal 24-OHase (days 1–7, P<0·01) and increased renal 1-OHase mRNA expression (days 1–5, P<0·01). LPD treatment failed to increase renal 1-OHase but did suppress 24-OHase mRNA expression (P<0·01) within 7 d of LPD treatment in adult rats. Renal expression of VDR mRNA decreased with age (P<0·001) and was suppressed by LPD treatment in both age groups (P<0·05) Feeding of adult rats with 10 d of LPD increased 1-OHase (P<0·05) and suppressed 24-OHase (P<0·001) as well as VDR (P<0·05) mRNA expression. These results indicate that the increase in serum 1,25(OH)2D3 level in adult rats during short-term LPD treatment is likely to be mediated by a decrease in metabolic clearance via the down-regulation of both renal 24-OHase and VDR expression. The induction of renal 1-OHase mRNA expression in adult rats requires longer duration of LPD treatment than in younger rats.

Effect of 1,25-dihydroxyvitamin D3 on rat pituitary prolactin release

1987 ◽  
Vol 116 (4) ◽  
pp. 459-464 ◽  
Author(s):  
Kid Törnquist
Keyword(s):  
Pituitary Cells ◽  
Dihydroxyvitamin D3 ◽  
Enhancing Effect ◽  
Hydroxyvitamin D ◽  
Dihydroxyvitamin D ◽  
Rat Pituitary ◽  
25 Hydroxyvitamin D ◽  
Pituitary Prolactin

Abstract. The effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on PRL secretion from rat pituitary in vivo and in vitro was investigated. Treating the rats for three days with 0.05 μg/kg per day had no effect on basal PRL secretion, whereas the TRH-induced PRL secretion was increased (P < 0.001). The enhancing effect of 1,25(OH)2D3 was blunted by verapamil. Incubating dispersed anterior pituitary cells with 10−8 mol/l 1,25(OH)2D3 induced a significant increase in PRL secretion after 96 h (364 ± 30 ng/well vs 481 ± 33 ng/well, P < 0.001; mean ± sem) compared with control cells. The TRH-induced PRL secretion was increased in cells incubated with 1,25(OH)2D3 for 144 h (0.766 ± 0.061 vs 1.024 ± 0.076 μg/well, P < 0.05; mean ± sem) compared with control cells. Neither 25-hydroxyvitamin D3 (25OH-D3) nor 24,25-dihydroxyvitamin D3 had any effects on the PRL secretion. However, when the cells were incubated with both 10−8 mol/l 1,25(OH)2D3 and 10−6 mol/l 25OHD3, the enhancing effect of 1,25(OH)2D3 on the basal PRL secretion was blunted. The results suggest that 1,25(OH)2D3 possibly affects the regulation of PRL release from the rat pituitary and that this effect is specific for 1,25(OH)2D3.

scholarly journals Vitamin D upregulates the macrophage complement receptor immunoglobulin in innate immunity to microbial pathogens

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Annabelle G. Small ◽  
Sarah Harvey ◽  
Jaspreet Kaur ◽  
Trishni Putty ◽  
Alex Quach ◽  
...  
Keyword(s):  
Vitamin D ◽  
Innate Immunity ◽  
Surface Expression ◽  
Microbial Pathogens ◽  
Cell Surface Expression ◽  
Complement Receptor ◽  
Hydroxyvitamin D ◽  
Dihydroxyvitamin D ◽  
Multistep Process ◽  
25 Hydroxyvitamin D

AbstractVitamin D deficiency remains a global concern. This ‘sunshine’ vitamin is converted through a multistep process to active 1,25-dihydroxyvitamin D3 (1,25D), the final step of which can occur in macrophages. Here we demonstrate a role for vitamin D in innate immunity. The expression of the complement receptor immunoglobulin (CRIg), which plays an important role in innate immunity, is upregulated by 1,25D in human macrophages. Monocytes cultured in 1,25D differentiated into macrophages displaying increased CRIg mRNA, protein and cell surface expression but not in classical complement receptors, CR3 and CR4. This was associated with increases in phagocytosis of complement opsonised Staphylococcus aureus and Candida albicans. Treating macrophages with 1,25D for 24 h also increases CRIg expression. While treating macrophages with 25-hydroxyvitamin D3 does not increase CRIg expression, added together with the toll like receptor 2 agonist, triacylated lipopeptide, Pam3CSK4, which promotes the conversion of 25-hydroxyvitamin D3 to 1,25D, leads to an increase in CRIg expression and increases in CYP27B1 mRNA. These findings suggest that macrophages harbour a vitamin D-primed innate defence mechanism, involving CRIg.

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