21,25-Dihydroxycholecalciferol. A metabolite of vitamin D3 preferentially active on bone

A vitamin D, metabolite in intestine more polar than 25-hydroxy-vitamin Vitamin D is believed to function in the regulation of calcium absorption from the gut by controlling the expression of genetic information (1, 2). Several groups hypothesize that vitamin D induces the synthesis of mRNA and a special protein(s) that comprises part or all of a calcium transport system in the intestinal mucosa cell. A calcium-binding protein induced by vitamin D has been isolated from the chick gut (3), and the vitamin has been shown to increase strikingly the Ca++-ATPase (4) and alkaline phosphatase (5) activity of intestinal microvillar membranes.Another important notion is that vitamin D must be metabolized prior to its functioning to induce calcium transport in the intestine. DeLuca and coworkers (6, 7) have isolated from blood a vitamin D3 metabolite which is 1.4 times as active as native vitamin D3. They determined the structure of this compound to be 25-hydroxy-vitamin D3(25-OH D3) and proposed that it was the active form of vitamin D3 (8, 9). Norman and associates (10) and later Kodicek and collaborators (11) used silicic-acid chromatography to detect a metabolite in chick intestine that was more polar than 25-OH D3. Haussler et al. (12) demonstrated that this metabolite was selectively associated with the nuclear chromatin of the target intestine and, more specifically, to a protein receptor molecule that was not a histone (13).In the present experiments, countercurrent distribution is employed to confirm the existence of a vitamin D3 metabolite in the intestine more polar than 25-OH D3. Isolation and biological assay of this metabolite reveals that it is the most efficacious and rapidly functioning metabolite of vitamin D3 yet tested and is, therefore, probably the active form of the vitamin in the chick intestine. Norman. Extraction techniquesLabeled steroids were administered dissolved in 1,2-propanediol. The chicks were killed by decapitation; the blood was collected with the aid of a funnel and the small intestine excised immediately. Intestinal-mucosa chromatin was prepared as described (12) and either the chromatin, whole intestine, or plasma was extracted with organic solvents by a modification of the procedure of Bligh and Dyer (14). The lipid extract was dried under N2 in preparation for countercurrent distribution. Countercurrent distribution (CCD) Distribution studies were carried out on an H.O. Post, model no. 2-B,100-tube automated machine; 100 transfers were performed with a 10-ml mobile (upper) phase and a 10-ml stationary (lower)phase. Solvent system 1: ethyl acetate-hexaneethanol-water 5:15:11:9 or 2: same solvents 5:15:7.5:12.5 were used.Metabolite from 1250 chick guts Triton X-100-washed chromatin (12) was isolated from the intestinal mucosa of all chicks 15-hr after an oral dose of 50 IU* of [3H]vitamin D3. The chromatin was washed with 10 mM Tris -HC1 (pH 7.5) to remove Triton, and then extracted with a total of 20 liters of acetone-dichloroethane 2:1 with the aid of a Waring blender. A...

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“…11 and manuscript in preparation). (21). They propose that this dihydroxy-metabolite is preferentially active on bone.…”

Section: Discussionmentioning

confidence: 99%

A Rapidly Acting Metabolite of Vitamin D ₃

Haussler

Boyce

Littledike

et al. 1971

Proc. Natl. Acad. Sci. U.S.A.

141

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“…Liquid-liquid partition column cochromatography of 1,000 dpm of the metabolite isolated in A (above) together with 600 dpm of pure 21,25-(OH)1D3 isolated by Suda et al (16).…”

Section: Deluca Manuscript In Preparation)mentioning

confidence: 95%

“…It is notable that this metabolite activates bone mineral mobilization and has slight activity in intestinal calcium transport (16). That the kidney has the highest concentration of 21,25-(OH)2D3 suggests a renal function for this compound ( Table 2).…”

Section: Deluca Manuscript In Preparation)mentioning

confidence: 99%

Regulation by Calcium of In Vivo Synthesis of 1,25-Dihydroxycholecalciferol and 21,25-Dihydroxycholecalciferol

Boyle

Gray

DeLuca

1971

Proc. Natl. Acad. Sci. U.S.A.

426

113

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A metabolite of vitamin D believed to be the metabolically active form in the intestine has recently been isolated in pure form from intestine and unequivocally identified in this laboratory as 1,25-dihydroxycholecalciferol (1,25-(OH)2D3)(1, 2). Simultaneously, Lawson et al. provided evidence for this structure with a partially purified material from kidney homogenates (3). This metabolite acts more rapidly than 25-(OH)D3 to initiate intestinal calcium transport (4-6). The kidney is the site of synthesis of 1,25-(OH)2D3 from 25-(OH)D3 (7); this observation was confirmed by Gray et al. (8). Other experiments have provided strong evidence that 1,25-(0H2)D3 is the metabolically active form of vitamin D in the intestine (9-11). It, therefore, seemed possible that the concentration of 1,25-(OH)2D3 in the serum and intestinal mucosa plays an important role in the adaptation of calcium absorption to the concentration of calcium in the diet (12).This report demonstrates that dietary calcium concentration has a profound effect on the in vivo production of 1,25-(OH)2D3 and 21,25-(OH)2D3. Furthermore, these alterations in metabolite balance correlate with changes in serum calcium, but not serum phosphate concentration. An independent study has also shown that dietary strontium has an equally marked effect on the production of 1, 325 pmoles of tritiated 25-(OH)D3 was administered intrajugularly in 0.05 ml of 95% ethanol with mild ether anethesia. The rats were fasted at this time, but water continued to be available ad libitum. 12 hr later the rats were killed by decapitation and blood serum was collected. The upper 50 cm of small intestine was quickly removed, flushed with ice-cold saline, slit open, and the mucosa was scraped off with a glass slide. The kidneys and livers were removed. The fore-and hind-limbs were stripped of muscle, the long bones were split, and the marrow was discarded. All tissues were stored at -16'C until they were extracted with chloroform and methanol (14). In early experiments, the tissue from 3 to 4 animals fed the same diet was combined before extraction, while in later experiments the serum calcium concentrations of the rats were analyzed for similarity before pooling of tissues. In other cases, the tissues from each animal were analyzed individually.Radioactive metabolites were separated on a 2 X 15 cm column containing 10 g of Sephadex LH-20 equilibrated with 35% Skellysolve B (petroleum ether, b.p. 67-680C) in 65% chloroform (15). The tissue extracts were applied to the column in 1 ml of the same solvent mixture and eluted with a further 190 ml. Recovery of radioactivity from the columns varied from 90 to 105%.The elution position of 1,25-(OH)2D3 on this column has been established (2, 8). The less-polar metabolite was identified as 21,25-(OH)2D3 by cochromatography. A sample of the 2131

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“…The mass spectrum of the tri(trimethylsilyl)derivative (III) and the mono(trimethylsilyl) derivative [MW = 488; structure II, produced from the tri(trimethylsilylated) compound by partial desilylation] and of the trimethylsilylether-diacetate exhibited an intense peak at m/e 131, which established the presence of a hydroxyl function at C-25 (2,11,12). In addition, the mass spectrum of the mono-(trimethylsilyl) compound (II, Fig.…”

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confidence: 99%

Identification of 1,25-Dihydroxycholecalciferol, a Form of Vitamin D3 Metabolically Active in the Intestine

2009

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A biologically active vitamin Ds metabolite ("peak V metabolite") more polar than 25-hydroxycholecalciferol has been isolated from chicken intestines in pure form as a mono(trimethylsilyl)ether derivative. The molecule has been identified as 1,25-dihydroxycholecalciferol by means of mass spectrometry, ultraviolet absorption spectrophotometry, and specific chemical reactions. It has been demonstrated (1) with tritiated vitamin D3 of high specific activity that vitamin D3 is converted to metabolites more potent and rapidly acting than the parent vitamin. The most abundant of these, 25-hydroxycholecalciferol (25-HCC) (2), acts more rapidly than vitamin D3 in enhancing both intestinal calcium transport and bone mineral mobilization (3). The existence in the intestine of additional polar metabolite(s) active in promoting calcium transport is evident from work in a number of laboratories (4-8). In agreement with Haussler et al. (8) and Myrtle et al. (9), we have observed that one of these metabolite(s), the peak V metabolite, induces intestinal calcium transport in deficient chickens more rapidly than vitamin D3 or 25-HCC, although it is less effective than 25-HCC in curing rickets or inducing bone mineral mobilization.By blocking the conversion of 25-HCC to peak V metabolite by prior administration of actinomycin D, we have shown (10) that peak V, or a further metabolite of peak V, is the metabolically active form of vitamin D in the intestine. In the presence of actinomycin D, the peak V compound stimulates intestinal calcium transport, whereas 25-HCC does not. This polar metabolite has now been isolated in pure form and identified as 1,25-dihydroxycholecalciferol (1,25-DHCC).ISOLATION 1450 vitamin D-deficient chicks were each given 2.5 jug of [1,2-3H]vitamin D3 (specific activity, 3.2 X 101 dpm/,ug), orally, in vegetable oil (Wesson). 24 hr later the chicks were killed. Methanol-chloroform extracts of the small intestines were prepared, and the chloroform extract was subjected to a sequence of chromatographic separations on Sephadex LH-20 and BioRad (beads) S-X8 (BioRad Labs., Richmond, Calif.). 11 jug of highly purified peak V metabolite was obtained. 8 jug of the metabolite was then reacted with 5 jul of TBT (a combination of TMS-imidazole, bis-TMSacetamide, and trimethylchlorosilane, Pierce Chemical Co., Rockford, Ill.) and 10 jul of pyridine for 10 min. The resulting tri(trimethylsilyl)ether derivative was chromatographed on a 1 X 60 cm column of Sephadex LH-20 in 50% chloroform in petroleum ether (bp 67-690C). The tri(trimethylsilyl) derivative was partially desilylated in 100 ul of 3.6 X 10-4 M HC1 in MeOH + 50 jul pyridine at 60°C for 4 hr. This gave a mixture of di(trimethylsilyl), mono(trimethylsilyl), and unsilylated products, which were separated by chromatography on Sephadex LH-20. The metabolite and the mono-(trimethylsilylated) metabolite were separately chromatographed in methanol on Sephadex LH-20 (11) before being used for structural identification.

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21,25-Dihydroxycholecalciferol. A metabolite of vitamin D3 preferentially active on bone

Cited by 134 publications

References 18 publications

A Rapidly Acting Metabolite of Vitamin D ₃

A Rapidly Acting Metabolite of Vitamin D ₃

Regulation by Calcium of In Vivo Synthesis of 1,25-Dihydroxycholecalciferol and 21,25-Dihydroxycholecalciferol

Identification of 1,25-Dihydroxycholecalciferol, a Form of Vitamin D3 Metabolically Active in the Intestine

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21,25-Dihydroxycholecalciferol. A metabolite of vitamin D3 preferentially active on bone

Cited by 134 publications

References 18 publications

A Rapidly Acting Metabolite of Vitamin D 3

A Rapidly Acting Metabolite of Vitamin D 3

Regulation by Calcium of In Vivo Synthesis of 1,25-Dihydroxycholecalciferol and 21,25-Dihydroxycholecalciferol

Identification of 1,25-Dihydroxycholecalciferol, a Form of Vitamin D3 Metabolically Active in the Intestine

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A Rapidly Acting Metabolite of Vitamin D ₃

A Rapidly Acting Metabolite of Vitamin D ₃