25-OHD3-26,23-LACTONE - 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. © 1979.