Excretion of Endogenous Steroids and Metabolites of [4‐¹⁴C]Pregnenolone in Bile of Female Rats

Gas‐liquid chromatography, gas chromatography‐mass spectrometry and radio‐gas chromatography were used to identify endogenous steroids and metabolites of [4‐¹⁴C]pregnenolone in bile from female bile fistula rats. About 70% of the administered radioactivity was recovered in the bile during the first 24 h, and less than 1% during the following three days. 76% of the biliary radioactivity was extracted and was separated according to conjugation. The free steroid fraction contained 2%, the glucuronide fraction 22%, the monosulphate fraction 45%, and the disulphate fraction 28% of this radioactivity. The following steroids were identified by gas chromatography‐mass spectrometry and were found to be labelled: glucuronide fraction: 3α‐hydroxy‐5α‐androstan‐17‐one, 3α‐hydroxy‐5α,17β‐pregnan‐20‐one and 3α,16α‐dihydroxy‐5α‐pregnan‐20‐one; monosulphate fraction: 3α,7α‐dihydroxy‐5α‐androstan‐17‐one and/or 3α,15α‐dihydroxy‐5α‐androstan‐17‐one, 3α,11β‐dihydroxy‐5α‐androstan‐17‐one and 3α,16α‐dihydroxy‐5α‐pregnan‐20‐one; disulphate fraction: 3α,7α‐dihydroxy‐5α‐androstan‐17‐one, 3α,11β‐dihydroxy‐5α‐androstan‐17‐one, 5α‐pregnane‐3β,20β‐diol and 3α,21‐dihydroxy‐5α‐pregnan‐20‐one. The main part of radioactivity in the steroid monosulphate fraction was associated with pregnenolone that had a very high specific activity. The amount of steroids excreted during the first 24 h after bile duct cannulation was about 700 μg, when no correction for losses was made. The large quantity of steroid metabolites excreted in the bile confirms the importance of this excretory pathway also for endogenous steroids. The quantitatively most important steroids were found to be unlabelled corticosterone metabolites: 3α‐ and 3β,11β,21‐trihydroxy‐5α‐pregnan‐20‐one and 3α,11β,15α,21‐tetrahydroxy‐5α‐pregnan‐20‐one. Several other unlabelled steroids were identified. The heterogenous distribution of radioactivity among the metabolites is in contrast to the more uniform labelling previously obtained with intact rats. This shows that the metabolism of injected labelled steroids in bile fistula rats is not representative of the conditions in the intact animal, where enterohepatic circulation results in a continuous labelling of precursor steroid(s).