Biological generation of tritiated vitamin D3 metabolites and their purification by high-performance liquid chromatography

Summary A simple, reproducible method for the biological synthesis of tritiated 24,25-dihydroxycholecalciferol, 25,26-dihydroxycholecalciferol and 1,25-dihydroxycholecalciferol is described. Kidney homogenates from both vitamin D deficient and replete chicks were usedin vitro to generate these dihydroxylated metabolites using 25 (23,24-³H) hydroxycholecalciferol as the substrate. Tritiated products were purified by Sephadex LH 20 chromatography followed by high-performance liquid chromatography; the identity of each metabolite was established by chromatography with authentic crystalline preparations.Presented at the Symposium organised by the Chromatography Discussion Group, held at Hatfield Lodge on 29 November 1979.