Biological activity of 1,25-dihydroxycholecalciferol.
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Biomedical subjects
Publications and source records attributed to H F DeLuca.
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The appearance of a polar metabolite of 25-hydroxychotecalciferol has been studied in the intestinal mucosas of nephrectomized rats and rats which have been made uremic by ureter ligation. In confirmation of previous work by Fraser and Kodicek, it was found that nephrectomy prevents the appearance of this metabolite in the intestine. On the other hand, equivalent amounts of the metabolite were found in uremic rats and in sham-operated controls, showing that the production of this metabolite is independent of the uremic state of the animals. In addition, it was shown that the metabolite produced in vitro by kidney homogenates is identical to that found in vivo in the intestine.
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Actinomycin D (or cycloheximide) administered prior to radioactive 25-hydroxycholecalciferol blocks the metabolism of 25-hydroxycholecalciferol to polar metabolites that accumulate in intestinal tissue, while it does not prevent the 25-hydroxylation of vitamin D(3) in the liver. Actinomycin D given after radioactive 25-hydroxycholecalciferol does not inhibit 25-hydroxycholecalciferol metabolism. These results indicate that 25-hydroxycholecalciferol must interact with the nuclei of cells to bring about the production of an enzyme(s) that converts it to its polar metabolites.
A biologically active vitamin D(3) 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.
The prior administration of actinomycin D prevents the metabolism of [(3)H]25-hydroxycholecalciferol to 1,25-dihydroxycholecalciferol, a metabolite of vitamin D(3) that is effective in the stimulation of intestinal calcium transport. In this paper, the question of whether the response of intestinal calcium transport to 1,25-dihydroxycholecalciferol is sensitive to actinomycin D was examined. While the response of intestinal transport to physiological amounts of 25-hydroxycholecalciferol is blocked by actinomycin D, the response of intestinal calcium transport to 1,25-dihydroxycholecalciferol is insensitive to the antibiotic. These results suggest that 1,25-dihydroxycholecalciferol, or a further metabolite thereof, is the metabolically active form of vitamin D in the intestine, that it functions by a process not involving transcription of DNA, and that the step sensitive to actinomycin D in the action of vitamin D on the intestine does not occur in the intestine, but is the conversion of 25-hydroxycholecalciferol to 1,25-dihydroxycholecalciferol in the kidney.
Tritiated 1,25-dihydroxycholecalciferol accumulates in several tissues, to an extent that varies with dietary calcium content, 12 hr after the administration of 325 pmoles of tritiated 25-hydroxycholecalciferol to rats. As the dietary and serum calcium concentrations increase, the amount of 1,25-dihydroxycholecalciferol is diminished and the concentration of 21,25-dihydroxycholecalciferol increases. This correlation is especially evident in rats given vitamin D(3). In vitamin D-deficient rats, the repression of 1,25-dihydroxycholecalciferol formation occurs with a diet containing 3% calcium and 20% lactose. The results suggest that the production of 1,25-dihydroxycholecalciferol, believed to be the metabolically active form of vitamin D in the intestine, is responsible for the adaptation of calcium absorption to low dietary concentrations of calcium.
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A simple yet powerful new chromatographic procedure for vitamin D(3) and its metabolites is described. Liquid-gel partition chromatography on Sephadex LH-20 using a solvent of various percentages of CHCl(3) in Skellysolve B (petroleum ether, bp 67-69 degrees C) permits excellent resolution of vitamin D(3), 25-hydroxyvitamin D(3), and their more polar metabolites. Of special importance is the resolution of the metabolites of vitamin D(3) more polar than 25-hydroxycholecalciferol. Because of this resolution, a new metabolite of vitamin D(3) has been demonstrated in the plasma of rats and in the intestines of chicks given 100 IU of vitamin D(3)-1,2-(3)H.