25-OHD3-26,23-lactone: a metabolite of vitamin D3 that is 5 times more potent than 25-OHD3 in the rat plasma competitive protein binding radioassay.

A new metabolite of Vitamin D₃ (25-OHD₃-26,23-lactone) has been found in the plasma of Vitamin D₃-toxic pigs and cows. This metabolite is at least 5 times more potent than 25-OHD₃ in the displacement of [³H]-25-OHD₃ from rat plasma protein binding sites under short-term incubation. This metabolite co-migrates with 24,25-(OH)₂D₃ on Sephadex LH-20 columns developed in chloroform:hexane 65:35 and with 25,26-(OH)₂D₃ on Sephadex LH-20 columns developed in hexane:chloroform:methanol 9:1:1. The presence of 25-OHD₃-26,23-lactone represents a possible contaiminant in the assay of 24,25-(OH)₂D₃ or 25,26-(OH)₂D₃ if only Sephadex LH-20 is used for pre-assay purification. 25-OHD₃-26,23-lactone is, however, resolved from 24,25-(OH)₂D₃ by high pressure liquid chromatography (HPLC) using Zorbax Sil silicic acid columns developed in either isopropanol:hexane 8:92 or isopropanol:methylene chloride 2.5:96.5. We assayed for the presence of this new metabolite of Vitamin D₃ and found it to be present in normal pig plasma and undetectable in normal cow plasma. Concentrations were elevated to 10–20 ng/ml following massive injection of Vitamin D₃ to both species.