1α-Hydroxyvitamin D3 (1α-OH-D3) is a synthetic sterol with biological characteristics similar to those of 1α, 25- dihydroxyvitamin D3 (1α, 25-(OH)2-D3), the apparent hormonal form of vitamin D. The synthetic sterol is virtually equipotent to the natural hormone, in vivo, and has been utilized recently to treat patients with defects in vitamin D metabolism. However, no information is presently available on its biochemical mode of action. In order to determine if 1α-OH-D3 functions by binding directly to target tissue receptors for 1α, 25- (OH)2-D3 or is first metabolized to 1α, 25-(OH)2-D3, we have carried out a detailed examination of the comparative biologic effects as well as the receptor binding properties of the two sterols. After a single oral dose of 162.5 pmol (2.5 IU) of either 1α-OH-D3 or 1α, 25-(OH)2-D3, rachitic chicks display equivalent increases in calcium absorption from the intestine; following a 2-3-hr latent period, both sterols elicit maximal effects at 9 hr and the responses undergo parallel decay between 24 and 72 hr. Comparison of the relative amounts of 1α-OHD3 and 1α, 25-(OH)2-D3 required to stimulate calcium absorption at 9 hr indicates that the sterols are equipotent in the dose range of 19.5-650 pmol (0.3-10 IU). In chronic administration studies, the synthetic sterol was slightly more antirachitic than 1α, 25-(OH)2-D3, with both sterols being 2-6 times more active than native vitamin D3. By contrast, in vitro studies utilizing competitive binding for the 1α, 25-(OH)2-D3 cytosol-chromatin receptor system from intestine demonstrate that 1α-OH-D3 binds to the receptor two-three orders of magnitude less avidly than 1α, 25-(OH)2-D3. Since the equipotency of these two sterols was not reflected at the molecular level, we performed the following experiment to show that 1α-OH-D3 is converted to 1α, 25-(OH)2-D3, in vivo. Rachitic chicks were given 1α-OHD3 and 3 hr later their intestinal chromatin was extracted. The suspected 1α, 25-(OH)2-D3 fraction was isolated by Celite chromatography and a saturating amount (26 pmol) of 1 α, 25- (OH)2-D3 was detected in the chromatin by competitive protein binding assay. Since 1α, 25-(OH)2-D3 was present in the chromatin receptor prior to the stimulation of calcium absorption by 1α-OH-D3, we conclude that 1α-OH-D3 is rapidly metabolized to 1α, 25-(OH)2-D3 and probably functions by conversion to the hormone. The conversion of 1 α-OH-D3 to 1α, 25-(OH)2-D3 was also observed in intestinal mucosa homogenates, in vitro, further verifying the occurrence of this enzymatic reaction.
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