Biosynthesis, purification and receptor binding properties of high specific radioactivity 1α,24(R),25-trihydroxy-[26,27-methyl-³H]-vitamin D₃

A procedure for the biosynthesis and purification of 1α,24(R),25-trihydroxy[26,27-methyl-³H]-vitamin D₃ (1,24,25-(OH)₃[³H]D₃) is reported. A kidney homogenate from chicks receiving a high calcium diet (3%) and oral supplements of 1α,25-dihydroxy vitamin d₃ (1,25-(OH)₂D₃) was used for C-24-hydroxylation of 1α,25-dihydroxy[26,27-methyl-³H]-vitamin F₃ (1,25-(OH)₂[³H]D₃), in vitro. Extraction and purification of the homogenate lipid fraction by Sephadex LH-20 and high performance liquid chromatography yielded radiochemically pure 1,24,25-(OH)₃[³H]D₃ with a specific radioactivity equivalent to the initial substrate (166Ci/mmol). The authenticity of the generated metabolite was assessed by comigration with synthetic 1,24,25-(OH)₃D₃ on high performance liquid chromatography and by equimolar competition with authentic radioinert 1,24,25-(OH)₃D₃ for binding to a purified receptor protein from rat kidney. Binding studies indicate the trihydroxylated metabolite competes 40-50% as effectively as 1,25-(OH)₂D₃ for hormone binding sites. Further analysis of l,24,25-(OH)₃D₃-receptor interaction reveals a high-affinity, saturable binding with an apparent K_d of 2.2 × 10^-9 M. These studies demonstrate that although slightly less active than 1,25-(OH)₂D₃, 1,24,25-(OH)₃D₃ is capable of hormone-like interactions, in vitro. The availability of this high specific radioactivity sterol should allow for clarification of its potential physiologic significance.