Evidence for a monoglucuronide of 1,25-dihydroxyvitamin D₃ in rat bile

We have isolated and characterized a monoglucuronide fraction of 9,10-secocholesta-5, 7, 10(19)triene-1α, 3β, 25-triol, 5,6-cis isomer (1,25-dihydroxyvitamin D₃) from rat bile. Polar radioactive metabolites of 1,25-dihydroxyvitamin D₃ were purified by a sequence of chromatographic procedures which utilized Amberlite XAD-2, diethylaminohydroxypropyl Sephadex LH-20, liquid-liquid partition on paper, and reverse phase chromatography on C-18 microparticulate columns. A purified radioactive substance showed maximal absorbance at 264 nm, indicating the presence of a triene in the 5,6-cis configuration. Mass spectrometry by fast atom bombardment of the product demonstrated an ion at m/z 637 atomic mass units that is consistent with a natriated sodium salt of a monoglucuronide of 1,25-dihydroxyvitamin D₃ ([MNa]Na⁺). Following methylation of the carboxylic acid group and formation of trimethylsilyl ethers of the hydroxyl groups, the fragmentation pattern of the product was compatible with that of a monoglucuronide of 1,25-dihydroxyvitamin D₃. The intact metabolite was treated with β-glucuronidase and the aglycon was isolated by chromatography on microparticulate silica. The aglycon co-migrated with authentic 1,25-dihydroxyvitamin D₃ during chromatography and it gave a mass fragmentation pattern consistent with 1,25-dihydroxyvitamin D₃. The aglycon was bound by an intestinal cytosol receptor with essentially the same affinity as 1,25-dihydroxyvitamin D₃. These findings indicate that bile contains a monoglucuronide of 1,25-dihydroxyvitamin D₃.