Biological activity of 25-hydroxycholecalciferol, a metabolite of vitamin D3.

In 1966 Lund and DeLu(al demonstrated unequivocally the existence of a major polar metabolite fraction of vitamin D in many tissues of rats given 3Hvitamin D3. This metabolite, which is able to cure rickets in rats at least as well as vitamin D, has since been demonstrated in human plasma,2 porcine plasma,3 and chicks.4 Further study revealed that this metabolite fraction, like vitamin D, initiates bone mobilization and intestinal transport of calcium,5 and its effect on calcium transport is more rapid than that of vitamin D. Stohs and DeLuca6 demonstrated that this metabolite fraction is virtually the exclusive form of vitamin D in intestinal nuclei prior to the initiation of biochemical events leading to the rise in calcium transport. Recently, the major if not the sole biologically active component of this fraction was isolated in pure form from the plasma of hogs fed vitamin D3, and its structure was unequivocally established as 25-hydroxycholecalciferol (25-OH D3).3 I The biological properties of this pure compound have now been examined in a number of systems, and the results are reported in this communication. Mllaterials and Methods.-Vitamin D: The crystalline vitamin D3 was obtained from General Biochemicals, Inc., Chagrin Falls, Ohio. 25-Hydroxycholecalciferol (25-OH D3) was isolated in pure form from the plasma of hogs, as described previously.3 The exact concentrations of these substances were determined by UV absorption at 265 mtz with a molar extinction coefficient of 18,200. The substances were dissolved in cottonseed oil for oral administration or in ethanol for intravenous administration. Preparation of rats: Weanling male albino rats were obtained from the Sprague-Dawley Co., or from the Badger Research Corporation, both of Madison, Wisconsin. They were housed individually in hanging wire cages and given food and water ad libitum. For the rickets cure test, we used the rachitogenic diet of Steenbock and Black8 as modified by the addition of crystalline vitamins at the level described by DeLuca et al.9 After the rats had been on this diet for 21 days, they were given either 25-OH D3 or vitamin D3 orally in 0.1 ml cottonseed oil or intrajugularly in 0.02 ml ethanol. Controls were given the appropriate carrier alone. The rats were killed 7 days later, their radii removed and stained with AgNO3 solution, and the degree of new calcification was scored visually as described in the U.S. Pharmacopoeia.10 In intestinal transport experiments, rats were fed for 5 weeks the adequate Ca and P diet described earlier,9 after which the animals exhibited a serum calcium concentration of 4.0-4.5 mg/100 ml. The 25-OH D3 or vitamin D3 was administered intravenously in 0.02 ml ethanol. At the indicated times, the ability of the duodenal section of intestine to transport calcium against a concentration gradient was assessed by the everted gut sac technique described earlier,1' except that the medium consisted of 125 mM NaCl, 10 mM fructose, 0.25 mM CaCl2 containing 45Ca, and 30 mM tris(hydroxymethyl)aminomethane buffer (Tris buffer), pH 7.4.12 The incubations were at 37?C under a stream of 02. Samples from inside and outside the sacs were taken after 1.5 hr and counted in a model 3000 Packard Tri-Carb liquid scintillation counter. The scintillator solution'2 consisted of 3 liters dioxane, 300 gm naphthalene, 14 gm 2,5-diphenyloxazole, 600 mg 1,4-bis-2-(4methyl-5-phenyloxazolyl) benzene, to which was added a solution of 36 mg Na2. EDTA in