Chemical lonization-mass spectrometric approach to structure determination of an intermediate in bile acid biosynthesis

Abstract

In the course of a study on the details of the biosynthesis of cholic acid from cholesterol, 5β-[26,27- 14C]cholestan-3α,7α,12α,24 S,25-pentol, an intermediate in the 25-hydroxylation pathway of cholic acid, was incubated for 2 min with the cytosolic fraction of rat liver homogenate in the presence of NAD. A precursor to cholic acid which appeared to be a ketone was isolate from the reaction mixture by thin-layer chromatography. This material proved to be of inadequate volatility for electron impact mass spectrometry and was therefore studied, without further purification, by techniques of chemical ionization mass spectrometry using ammonia as the reagent gas. The spectrum was rerecorded using argon mixed with ammonia to induce additional fragmentation. One of these fragments corresponded to a McLafferty rearrangement of a 24-keto-25-hydroxycholestane derivative. To obtain additional evidence for this structure the following sequence of reactions was conducted on about 20 μg of the intermediate: (1) periodic acid oxidation, (2) diazomethane treatment, and (3) chromic acid oxidation. The change in molecular weight after each reaction agreed with the presence of a 25-hydroxy-24-keto side chain and three secondary hydroxyl groups in the molecule. Therefore, it could be deduced that the intermediate was 3α,7α,12α,25-tetrahydroxy-5β-cholestan-24-one. This work demonstrates that chemical ionization-mass spectrometic techniques can be a labor-saving alternative to other methods of structure determination and that 3α,7α,12α,25-tetrahydroxy-5β-cholestan-24-one is probably an intermediate in the 25-hydroxylation pathway of cholic acid from cholesterol.