Metabolism Of 25-hydroxycholecalciferol Research Articles

Regulation of 25-hydroxycholecalciferol metabolism by inorganic phosphate.

Regulation of 25-Hydroxycholecalciferol Metabolism by Inorganic Phosphate Nutrition Reviews, Volume 31, Issue 6, June 1973, Pages 188–190, https://doi.org/10.1111/j.1753-4887.1973.tb05175.x Published: 01 June 1973

Identification of 8α,25-dihydroxy-9,10-seco-4,6,10(19)-cholestatrien-3-one as a product of metabolism of 25-hydroxycholecalciferol by liver microsomes

The ability of liver microsomes, sites of synthesis of 25-hydroxycholecalciferol, to further metabolize 25-hydroxycholecalciferol has been assessed. When liver microsomes were incubated with 25-hydroxycholecalciferol in the presence of cytosol, a metabolite was isolated that comigrated with 8α,25-dihydroxy-9,10-seco-4,6,10(19)-cholestatrien-3-one in three different chromatographic systems. The ultraviolet spectrum (220–350 nm) and mass spectrum of the purified metabolite were identical to that of synthetic 8α,25-dihydroxy-9,10-seco-4,6,10(19)-cholestatrien-3-one. This study indicates that liver microsomes convert 25-hydroxycholecalciferol to 8α,25-dihydroxy-9,10-seco-4, 6,10(19)-cholestatrien-3-one. The significance of this metabolite, which has been shown previously by others to be produced by alveolar macrophages, has yet to be determined.

Calcium-dependent metabolism of 25-hydroxycholecalciferol in silver eel tissues

We investigated the in vitro metabolism of [26,27 3H]-25-(OH)D 3 in different eel tissues. After incubation with [ 3H]-25-(OH)D 3, tissues were extracted with methanol-chloroform and chromatographed on Sephadex LH 20 columns. Two derivatives less polar than 25-(OH)D 3 were detected, the first one being sensitive to KOH treatment. Three peaks more polar than 25-(OH)D 3 were also found: peak I migrated close to the 24,25-(OH) 2D 3 area and was quantitatively the most important, but the presence of 24,25-(OH) 2D 3 could not be demonstrated; peak II migrated in the 1,25-(OH) 2D 3 region; and peak III had an elution position twice that of peak II. After 6-h incubation of tissues isolated from control eels, peak I was found in all tissues including intestine and gills. It was highest in pituitary gland and brain and lowest in ovaries and muscle. It was not significantly modified 20 days after ablation of the corpuscules of Stannius. In contrast, in vivo daily calcium chloride injection was followed 24 hr later by a significant increase in the [ 3H]-25-(OH)D 3 conversion into peak I in gills, intestine, and the spinal cord and by an inhibition of this conversion in pituitary gland, skin, and muscle. The inhibition was found in all tissues after five daily calcium injections. Calcium injection had no effect on the in vitro metabolite synthesis by the corpuscules of Stannius. These results suggest that vitamin D is not metabolized in the same way in eel as in mammals and that this metabolism could in part be calcium dependent.

Metabolism of 25-hydroxycholecalciferol in human promyelocytic leukemia cells (HL-60). Isolation and identification of (5Z)- and (5E)-19-nor-10-oxo-25-hydroxycholecalciferol.

Human promyelocytic leukemia cells incubated with 25-hydroxy[26,27-methyl-3H] cholecalciferol (1 microCi) or non-radioactive 25-hydroxycholecalciferol (550 micrograms) produced significant quantities of two vitamin D3 metabolites. The two metabolites were isolated and purified by methanol chloroform extraction and a series of chromatographic procedures. The metabolite purification and elution positions on these columns were followed by radioactivity and their ultraviolet absorption at 310 nm. The two metabolites have been unequivocally identified as (5Z)- and (5E)-19-nor-10-oxo-25-hydroxycholecalciferol by ultraviolet absorption spectrophotometry, mass spectrometry, Fourier-transform infrared spectrophotometry and co-chromatography with synthetic compounds on a high-performance liquid chromatograph. (5E)- but not (5Z)-19-nor-10-oxo-25-hydroxycholecalciferol was able to induce HL-60 cell phenotypic and functional differentiation. However, these two metabolites of 25-hydroxycholecalciferol did not bind specifically to the chick intestinal 3.7 S. receptor protein for 1 alpha,25-dihydroxycholecalciferol. The precise biological role of these metabolites is as yet unclear.

Metabolism of 25-hydroxycholecalciferol in a teleost fish, the rainbow trout ( Salmo gairdneri)

Plasma concentrations of vitamin D 3 (cholecalciferol) metabolites have been studied in rainbow trout ( Salmo gairdneri) adapted to varying environmental calcium concentrations in both fresh water and artificial seawater, and in natural seawater. In vivo, intraarterial injection of tritiated 25-hydroxycholecalciferol was followed by its transformation to a number of metabolites including compounds that cochromatographed on high performance liquid chromatography (HPLC) with 1,25-dihydroxycholecalciferol and 25,26-dihydroxycholecalciferol. Hypercalcaemia and increased environmental calcium were associated with a greater transformation to the compound cochromatographing with 25,26-dihydroxycholecalciferol, while hypocalcaemia and reduced environmental calcium concentrations induced more conversion to the 1,25-dihydroxycholecalciferol-like compound. In vitro, both metabolites were produced by liver but not by kidney preparations, and the difference in conversion ratios observed in vivo associated with changes in plasma calcium were also seen in vitro. It is concluded that the metabolism of 25-hydroxycholecalciferol in the trout can be influenced by calcium status, but at present the physiological importance of this metabolism and the mechanisms and site(s) of action of the metabolites are unknown.

In vitro metabolism of 25-hydroxycholecalciferol by isolated cells from human decidua.

An increase in maternal serum levels of 1 alpha, 25-dihydroxycalciferol during pregnancy has been linked to enhancement of intestinal calcium absorption. Several sites of its synthesis have been proposed in different species, human decidua being one of them. Collagenase-dispersed decidual cells isolated from term placenta were fractionated on a Percoll gradient. The isolated cells were set in culture in the presence of 6 nM [3H]cholecalciferol. Two cell populations of similar morphology hydroxylated the substrate, yielding a compound that had a mass spectrum identical to and that comigrated with authentic 1 alpha, 25-dihydroxycholecalciferol in four chromatographic systems and bound to a specific rachitic chick receptor. These preparations, thus, provide a potential system by which the kinetics and regulation of the synthesis of the hormonal form of vitamin D by human placenta can be studied in vitro.

Effects of hypophysectomy and growth hormone treatment on renal hydroxylation of 25-hydroxycholecalciferol in rats.

Growth hormone stimulates intestinal calcium absorption. This action has been linked to vitamin D metabolism. We have investigated the effects of hypophysectomy and GH treatment on renal metabolism of 25-hydroxycholecalciferol (25-OH-D3). Renal hydroxylation of 25-OH-D3 was measured in vitro using the renal slice technique. Experiments were performed in young F344 rats fed a vitamin D-replete, low calcium diet for 4 weeks. In hypophysectomized rats, renal conversion of 25-OH-D3 to 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) was markedly reduced compared with sham-operated rats. Renal conversion of 25-OH-D3 to 24,25-(OH)2D3 was markedly increased in hypophysectomized rats compared with sham-operated rats. Treatment of hypophysectomized rats with rat GH (rGH) for 10 days resulted in a significant increase in renal conversion of 25-OH-D3 to 1,25-(OH)2D3 and a significant decrease in conversion to 24,25-(OH)2D3. Rat GH treatment caused no significant changes in serum levels of immunoreactive parathyroid hormone. Serum calcium concentrations were similar in all groups, and serum phosphorus was low in hypophysectomized rats. Treatment of hypophysectomized rats with ovine GH for 6 days caused changes which were much less pronounced than those induced by rGH. Renal conversion of 25-OH-D3 to 1,25-(OH)2D3 and 24,25-(OH)2D3 correlated well with growth rate (weight gain). These results suggest that GH, either directly or indirectly, modulates renal metabolism of 25-OH-D3.

Metabolism of 25-hydroxycholecalciferol by human bone.

Although there is abundant evidence from animal studies that 1,25-dihydroxychole-calciferol [1,25-(OH)2D3] is synthesized in the kidney, it is still unknown whether human bone might also be involved in the formation of this metabolite. To investigate whether 1,25-(OH)2D3 can be synthesized by human bone, 25-hydroxycholecalciferol (25-OH D3) metabolism was assessed in vitro by incubation with human trabecular bone. The bone tissue, obtained from the femoral heads of 34 patients undergoing hip replacement surgery, was ground and thoroughly cleaned of blood and bone marrow. Five hundred mg of spongiosa were incubated with (3H)-25-OH D3 at a concentration of 1.67 nM in 2 ml Eagle’s minimum essential medium. The metabolites formed were separated either by LH 20 column chromatography followed by high performance liquid chromatography (HPLC) or by HPLC alone and the elution profiles compared to either commercially available or biologically synthesized tritiated 1,25-(OH)2D3 and 24,25-(OH)2D3. The formation of a metabolite which was chromatographically similar to 1,25-(OH)2D3 was observed in 15, and a metabolite with the chromatographic features of 24,25-(OH)2D3 in 13 of 21 bone samples. In 4 samples an additional metabolite more polar than 1,25-(OH)2D3 was observed. I n 6 bone samples no chromatographic evidence for the production of any metabolite of 25-OH D3 could be found. Ten per cent isopropanol, 1 ml of uremic serum or replacement of NADPH by NADH in the incubation medium completely suppressed the production of all derivatives. The demonstration that human bone is able to metabolize 25-OH D3 may be important for the understanding of bone physiology.

Metabolism of 25-hydroxycholecalciferol after renal transplantation

Metabolism of 25-hydroxycholecalciferol after renal transplantation

Metabolism of 25-hydroxycholecalciferol by kidney cell lines

The metabolism of 25-hydroxyvitamin D 3 to 24,25-dihydroxyvitamin D 3 by the rhesus monkey cell line LLC-MK 2 and similar reactions of the African green monkey cell lines, Vero and BSC-1, were observed. In addition, a second metabolite produced by the former cell line was identified as 25,26-dihydroxyvitamin D 3. Four other kidney cell lines, canine MDCK, bovine MDBK, embryonic bovine MA-115, and rabbit RK-13 produced only trace amounts of metabolites, and baby hamster kidney cells BHK-21 and owl monkey kidney cells OMK-21 synthesized no metabolites of 25-hydroxyvitamin D 3. Suspension cultures of LLC-MK 2 and Vero were found to produce oxidation products linearly for up to 3 h with specific activities of 5.23 and 2.40 pmol/h mg protein, respectively.

The regulation of 25-hydroxycholecalciferol metabolism in the kidney of Japanese quail ( Coturnix coturnix japonica) by sex hormones and by parathyroid extract

The effect of sex hormones and of parathyroid extract (PTE) of the metabolism of 25-hydroxycholecalciferol (25-HCC) in the kidney of Japanese quail was studied in vitro. Production of 1,25-dihydroxycholecalciferol (DHCC) was stimulated by oestrogen and by PTE and these effects were additive; the action of androgen was to inhibit the 1-hydroxylase. Production of 24,25-DHCC was inhibited by oestrogen and by PTE but androgen had no significant effect. A marked synergism between oestrogen, androgen, and PTE was shown: when all three hormones were administered together a striking increase occurred in 1,25-DHCC production compared with all other hormone treatments. Drugs that inhibited ovulation and shell calcification inhibited the amount of 1,25- relative to 24,25-DHCC produced by the kidney homogenates. These results are discussed in relation to the control of calcium metabolism in laying birds and it is suggested that (a) reproductive steroids are mainly responsible for the long-term increase in calcium absorption that occurs in birds coming into lay by increasing the amount of renal 25-HCC-1-hydroxylase; (b) parathyroid hormone is responsible for the short-term increase in the activity of the renal 1-hydroxylase during the egg cycle.

Metabolism of 25-hydroxycholecalciferol in human bone.

Metabolism of 25-hydroxycholecalciferol in human bone.

Evidence for a direct effect of parathyroid on the metabolism of 25-hydroxycholecalciferol by chick kidney cells in vitro

1. 1. The production of 1,25 hydroxycholecalciferol (1,25-(OH) 2D 3) by dispersed kidney cells obtained from vitamin D or calcium deficient, and from vitamin D-fed or repleted birds, was evaluated. 2. 2. Once stimulated in vivo by vitamin D or calcium deficiency, the kidney cells continued the accelerated production of 1,25-(OH) 2D 3 for 3 hr, in vitro. 3. 3. Pure bovine parathyroid hormone and a homogenate, extract or TCA powder of chick parathyroid gland (PTG) increased significantly the production of 1,25-(OH) 2D 3 by kidney cells. 4. 4. The effect of avian PTG material on 24,25-(OH) 2D 3 (24,25-(OH) 2D 2) production was not significant. 5. 5. The effect of PTG homogenate was independent of calcium concentration in the incubation medium. A high calcium concentration (3.0 mM) somewhat reduced the formation in vitro of 1,25-(OH) 2D 3 but stimulated slightly the production of 24,25-(OH) 2D 3. 6. 6. The effect of parathyroid sources was significant in cells taken from vitamin D replete and not vitamin D deficient birds.

Lack of effect on ovariectomy on the metabolism of vitamin D and intestinal calcium-binding protein in female rats.

The metabolism of 25-hydroxycholecalciferol (25-(OH)D3), plasma concentration of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) and the amount of calcium-binding protein (CaBP) in duodenal mucosa were determined in ovariectomized rats and were compared with data observed in normal age-matched cyclic rats. Sephadex LH-20 and high-pressure liquid chromatography were used for the study of the metabolism of 25-(OH)D3. The concentration of 1,25-(OH)2D3 in plasma and prolactin in serum were measured by radioimmunoassay. Calcium-binding protein in duodenal mucosa was determined immunologically using electroimmunodiffusion. The results showed that the lack of ovarian hormones and low prolactin levels observed in ovariectomized rats did not promote a significant change in the metabolism of 25-(OH)D3, in the levels of 1,25-(OH)2D3 in the circulation or in the amount of CaBP in duodenal mucosa. It is possible that the regulation of 25-(OH)D3 by sex hormones is restricted to the state of calcium stress such as during egg-laying in birds or pregnancy and lactation in mammals.

The In Vitro Production and Activity of 24, 25-Dihydroxycholecalciferol in Cartilage and Calvarium

Previously reported results from our laboratory have indicated that 24, 25-dihydroxycholecalciferol can be formed in vitro during incubations of cartilage tissue or cartilage cells with 25-hydroxycholecalcified. They have also demonstrated the high potency of this dihydroxymetabolite of vitamin D3 in stimulating in vitro the sulfate incorporation into proteoglycans of cartilage cells in culture and in decreasing in vitro the parathyroid hormone action on rat calvarium phosphatases activities. The present report shows that 24, 25-)OH)2 D3 can also be produced during rat calvarium incubations with 25-hydroxycholecalciferol and that therefore calvarium as well as cartilage might be both a site of formation and a site of action for 24, 25-(OH)2 D3. The review of recent experimental and clinical investigations strongly suggests that this metabolite has a physiological significance and may be specifically active on some parameters of bone mineralization. Further studies on the cartilage and calvarium abilities to convert 25-hydroxycholecalciferol into 24, 25-(OH)2 D3 shows that this transformation occurs in the mitochondrial fraction but that it does not seem modified by factors known to control the 25-hydroxycholecalciferol metabolism in the kidney. Finally the analysis of experimental and clinical results published so far does not yet bring enough information to understand the significance of this extrarenal metabolism of 25-hydroxycholecalciferol.

Effect of oestrogen and 1,25-dihydroxycholecalciferol on 25-hydroxycholecalciferol metabolism in primary chick kidney-cell cultures.

Primary cultures of chick kidney cells convert 25-hydroxycholecalciferol into more-polar metabolites. Cells from vitamin D-deficient chicks have high 25-hydroxycholecalciferol 1 alpha-hydroxylase (1 alpha-hydroxylase) activity, but no 25-hydroxycholecalciferol 24-hydroxylase (24-hydroxylase) activity. Physiological concentrations of 1,25-dihydroxycholeclaciferol suppress 1 alpha-hydroxylase and induce 24-hydroxylase activity. The inhibition of 1 alpha-hydroxylase preceded the induction of 24-hydroxylase. In contrast, oestradiol-17 beta had no effect on the activity of either hydroxylase under a variety of experimental conditions. These results clearly demonstrate that 1,25-dihydroxycholecalciferol, but not oestrogen, acts directly on the kidney cells to regulate the metabolism of 25-hydroxycholecalciferol.

The in vitro metabolism of 25-hydroxycholecalciferol by pig kidney: effect of low dietary levels of calcium and phosphorus.

1. Homogenates of pig kidney incubated in vitro convert 25-hydroxycholecalciferol to the dihydroxymetabolites almost as efficiently as homogenates of chick kidney under similar conditions. 2. The cortex of the kidney carries out this conversion more efficiently than does the medulla. 3. Kidneys from pigs given low calcium or low phosphorus diets produce about 40% more 1,25-dihydroxycholecalciferol than those from pigs given diets containing normal levels of Ca and P.

25-Hydroxycholecalciferol metabolism in hypophysectomized rats.

The metabolism of 25-hydroxycholecalciferol (25-(OH)Da) was investigated in vitamin D normal rats, 4 weeks after hypophysectomy. Compared to sham-operated controls, these animals were hypophosphatemic and their parathyroid glands were significantly smaller than normal. Eighteen hours after ip injection of a single dose of tritiated 25-(OH)D3 (50 pmol), chromatography of serum extracts on Sephadex LH-20 and high pressure liquid chromatography showed that the percentage of radioactivity corresponding to 24,25-dihydroxycholecalciferol [24,25-(OH)2D3] was significantly higher in hypophysectomized rats (11.7 ± 0.95% vs. 6.9 ± 0.78%). No radioactivity was detected in the area corresponding to 1,25-dihydroxycholecalciferol. In hypophysectomized rats injected 2 weeks with radioactive 25-(OH)D3 the amount of 24,25(OH)2D3) was still higher than in sham-operated controls. Parathyroidectomy 48 h before the injection of [2H]25(OH)D3 abolished the difference between the two groups. The percentage of 24,25-(OH2D3t in th...

Vitamin D and cartilage. I. In vitro metabolism of 25-hydroxycholecalciferol by cartilage.

In the present work, the capacity of cartilage to metabolize 25-hydroxycholecalciferol was investigated. Cartilage preparations from growth plate, articular surface, rib, scapula, and ear were isolated from 3-week-old normal rabbits and chickens. Each tissue was separately incubated with tritiated 25-hydroxycholecalciferol (, x 10(-9) M) for 1-24 h. Incubations of kidney and muscle were performed simultaneously for comparison. Similarly, cultured chondrocytes isolated from rabbit growth plate and articular cartilage were incubated for 1 or 20 h in medium free of fetal calf serum. After methanol-chloroform extraction of tissues, cells, and their respective media, chloroform phases were chromatographed on Sephadex LH-20 columns. The results show that kidney and cartilage are able to convert 25-hydroxycholecalciferol into a derivative which migrates in the 24,25-dihydroxycholecalciferol region. Cartilage tissue previously boiled is unable to metabolize 25-hydroxycholecalciferol. The conversion of 25-hydroxycholecalciferol occurs with all types of cartilage and is also observed in incubations of cultured chondrocytes. In the latter, the polar 25-hydroxycholecalciferol derivative is detected as early as 1 h after addition of 25-hydroxycholecalciferol. Two findings suggest that the polar derivative of 25-hydroxycholecalciferol produced by cartilage is 24,25-dihydroxycholecalciferol: 1) the cartilage derivative and 24,25-dihydroxycholecalciferol (synthetic and biosynthetic) comigrate during Sephadex LH-20 and high liquid pressure chromatography; and 2) both the cartilage derivative and 24,25-dihydroxycholecalciferol are sensitive to periodate treatment.

Metabolism of 25-Hydroxycholecalciferol and 1α-Hydroxycholecalciferol in Experimental Rat with Chronic Renal Failure

Metabolism of 25-Hydroxycholecalciferol and 1α-Hydroxycholecalciferol in Experimental Rat with Chronic Renal Failure