A Rapidly Acting Metabolite of Vitamin D ₃

Abstract

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...

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

“…than 25-(OH)D3 to initiate intestinal calcium transport (4)(5)(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).…”

Vitamin D

“…They concluded that this material, which they called peak 5, was formed in the intestinal nuclei and other target cells from 25-hydroxycholecalciferol. This polar compound became of great interest when Kodicek et al (1970), Haussler et al (1971 and Myrtle & Norman (1971) showed that it was more active than D3 in producing increased intestinal absorption of calcium. A major advance occurred very shortly afterwards when Fraser & Kodicek (1970) established conclusively that the kidney was the unique site of biosynthesis of this biologically active vitamin D metabolite.…”

The Stimulation of 1,25-Dihydroxycholecalciferol Metabolism in Vitamin D-deficient Rats by 1,25-Dihydroxycholecalciferol Treatment

“…When given intravenously as a single-pulse dose, 1,25-(OH)2D3 has been shown to act more rapidly than 25-OHD3 in stimulating intestinal calcium transport (11,12) and in activating the bone calcium mobilization system (12,13). Although more active than 25-OHD3 in the intestinal calcium transport system (11-13), 1,25-(OH)2D3 was equally as active as 25-OHD3 in the bone calcium mobilization response and was much less active in the cure of rickets in rats (12).…”