Bile acids are synthesized from cholesterol in the liver excreted into duodenum via the bile duct as their glycine and taurine conjugates, and converted in part into secondary bile acids by intestinal bacteria. It is well-known that further hydroxylation at C-1, -2, -4, -6 and -19, and conjugation at C-3 with glucuronic acid or sulfuric acid take place in the liver. In this paper bio-analytical studies on bile acids by hyphenated mass spectrometry related to drug metabolism will be discussed.
In the first, to clarify an extrahepatic reduction system, 18O labeled 3-oxo bile acids were synthesized and after incubation with human blood, biotransformed products were separated and characterized by GC-MS. The 3-oxo group was reduced into the 3α- and 3β-hydroxyl function catalyzed by the enzyme (s) in human red blood cells. This result strongly implies that biologically active compounds such as drugs with the oxo group are also transformed into hydroxylated metabolites in human blood.
On the biosynthesis of bile acids, the final step involving the oxidative cleavage of a side chain of 5β-cholestanoic acid, which has a chiral center at C-25, to form primary bile acids takes place in the peroxisomes. From a stereochemical point of view, the dehydrogenation mechanism of the biotransformation of 5β-cholestanoic acid into (24E)-5β-cholest-24-enoic acid was then studies with GC-MS. Substrates labeled with 2H at C 24 and C-25 stereoselectively were incubated with a rat liver light mitochondrial fraction and the stereospecific elimination of a pro-R hydrogen at C-24 in both (25R) and (25S)-cholestanoic acids indicating syn-elimination for the former, whereas anti-elimination for the letter was observed. When CoA thioesters of (25R)-and (25S)-5β-cholanoic acids were incubated individually with a rat liver peroxisomal fraction, rapid epimerization to form an equal mixture of stereoisomers from either direction was occurred. Moreover only the 25S antipode was easily transformed into dehydrogenated 5β-cholestanoic acid by peroxisomal acyl-CoA oxidase.
The conjugation of carboxylic acids with n-glucuronic acid to form acyl glucuronides plays a significant role in the metabolism and disposition of drugs. The formation of 24-acyl glucuronides of bile acids in the rat liver are also discussed.
