Figure 2  .

HPLC profile of synovial fluid fibroblast (A) [³H]-25-(OH)D₃ and (B) [³H]-1,25-(OH)₂D₃ metabolism. HPLC conditions are as described in Methods. The dotted traces show the positions of known tritiated forms of 25-(OH)D₃ (peak A), 24,25-(OH) ₂D₃ (peak B), and 1,25-(OH) ₂D₃ (peak C). As no radioactive tracer was available, the expected position of a tritiated 1,24,25-(OH) ₃D₃ peak was calculated using an ultraviolet trace of unlabelled markers in conjunction with the radioactive trace above. The solid trace in figure 2 (A) shows the metabolism of substrate [³H]-25-(OH)D₃ to its 24-hydroxylated form, [³H]-24,25-(OH)₂D₃, by synovial fluid fibroblasts pre-treated for 24 hours with 10^-8 M 1,25-(OH) ₂D₃. The position of an unidentified product, which was more polar than [³H]-24,25-(OH) ₂D₃, is indicated by peak D. The solid trace in figure 2 (B) shows the metabolism of substrate [³H]-1,25-(OH)₂D₃ by synovial fluid fibroblasts pre-treated for 24 hours with 10^-11 M 1,25-(OH)₂D₃. A metabolite with an elution time representing that of [³H]-1,24,25-(OH)₃D₃ is shown by peak E and the position of an unidentified product, which was more polar than [³H]-1,24,25-(OH)₃D₃, is indicated by peak F; dpm = disintegrations per minute.