Vitamin D has been called "the most dominating single factor in the regulation of the absorption of calcium" (1). Although experiments with segments of small intestine in vitro have demonstrated that the vitamin is required to maintain an active transport mechanism for calcium in the mucosa (2-4), the molecular events underlying the action remain unknown. To define them, detailed information on the metabolism of vitamin D, now lacking, is needed.The metabolic fate of the vitamins D can be studied most effectively with radioactive compounds, for chemical methods of estimation are relatively insensitive or nonspecific, and biological assay is tedious and difficult. C14-vitamin D3 labeled at C3 (5) and randomly labeled C14-vitamin De (6) have been prepared by prior investigators, and with the latter material Kodicek and collaborators have provided much of the available metabolic data (7-10). The information remains limited, however, partly because it is difficult to prepare sufficient quantities of material of relatively high specific activity. Accordingly, the present studies were begun to develop more efficient methods for the preparation of radioactive vitamin D for metabolic studies. Tritium-labeled vitamin D3 and C14-labeled vitamin D, have been prepared, and the major pathways of transport and metabo-* Submitted for publication October 28, 1963; accepted December 26, 1963. Supported by U. S. Public Health Service grant AM-01483.Portions of this work were presented at the 55th Annual Meeting of the American Society for Clinical Investigation, Atlantic City, N. J., April 1963. lism in the rat identified (11). Vitamin D is absorbed chiefly into lymph, distributed widely in the tissues but accumulated in highest concentration in liver, and excreted mainly into bile. The present report describes in detail the preparation of the radioactive compounds and their intestinal absorption in the rat.
Materials and MethodsPreparation of H'-vitamnin D,. Initial experiments demonstrated persistence of biological activity in a solution of vitamin D, in 80% acetic acid after 16 hours at 230 C in an atmosphere of N2. Therefore 1 g of crystalline vitamin D, under N, 1 was labeled 2 by catalytic exchange in tritiated acetic acid under identical conditions (schedule TR-1). The product, a brownish material in a vial under N,, was applied for thin-layer chromatography on plates of activated silicic acid gel 3 0.5 mm thick. Reference vitamin D, was applied to the outer margins. The plates were developed in the dark for 45 minutes with CHC13 as the solvent, dried briefly at room temperature, sprayed lightly with 0.001%o Rhodamine 6 G in acetone, and viewed under an ultraviolet lamp (maximal emission, 253.7 mA&) to localize the dark, absorbing band of vitamin D3. The bands of gel were scraped from the plates and eluted repeatedly with CHC13. Vitamin D in the eluate, and generally in the present studies, was estimated chemically with an SbCl, reagent (12). (Dried samples were treated with the SbCl3 reagent at room temperature for e...
