Conjugated forms of [3H] alpha, 25-dihydroxyvitamin D3 in rat bile

When small doses of [3H]D3, [3H]25-OHD3 and [3H]alpha, 25-diOHD3 were administered intravenously to rats 6.3 +/- 1.1% (means +/- SEM, n = 4), 9.7 +/- 0.9% (n = 6) and 12.8 +/- 2.6% (n = 8), respectively, of the administered radioactivity was excreted in bile. The radioactive biliary conjugated metabolites were analysed by ion exchange chromatography: in the case of all 3 substrates about 30% of metabolites were found to be cationic on the basis of their being retained on sulphopropyl-Sephadex G-25 (H+-form) when applied in 70% methanol. The balance of the metabolites were neutral and anionic and were analysed on TEAP-Lipidex: in the case of 1 alpha, 25-diOHD3 the following metabolite classes were detected (on the basis of co-elution with authentic standards) (in order of quantitative importance): taurine conjugates, neutral metabolites, monosulphates, glucuronides, carboxylic acids, glycine conjugates and disulphates. Alkaline hydrolysis of the taurine and glycine conjugates yielded products 60% of which now chromatographed as carboxylic acids. Hydrolysis of the glucuronide and monosulphate fractions indicated significant levels of mixed conjugation yielding some products which now chromatographed as glycine and taurine conjugates, respectively. The nature of the cationic conjugates was not elucidated but they had the following properties: they could be hydrolysed by alkali to yield non-cationic radioactive metabolites (these released metabolites were heterogeneous as judged by TEAP-lipidex chromatography); they were partially hydrolysed to non-cationic forms by beta-glucuronidase; and on reverse-phase HPLC they had an elution profile that was significantly different to that of histidyl-, ornithyl- or lysyl-calcitroic acid.