The nature of urinary bile acid conjugates in patients with extrahepatic cholestasis

Abstract

Urinary bile acids from patients with extrahepatic cholestasis were extracted with Sep-pak C₁₈ cartridges and group separated on diethylaminohydroxypropyl Sephadex LH-20. The nature of the different conjugates of cholic and chenodeoxycholic acid in the fractions was studied after further separation by preparative thin-layer chromatography.

The free and glycine-conjugated bile acids were quantified by capillary gas chromatography and identified by gas chromatography-mass spectrometry (GC/MS). Taurine conjugates were split with cholylglycine hydrolase and the liberated free bile acids analysed by GC/MS. Sulphate esters were hydrolysed with Helix pomatia and the resulting bile acid derivatives were analysed as above. After hydrolysis with cholylglycine hydrolase, the glucuronides of the unconjugated bile acids were separated and identified by GC/MS.

Amino acid analysis of the different fractions revealed that glycine and taurine were the only amino acids present in connection with cholic and chenodeoxycholic acid. Large amounts of monosulphated bile acid conjugates were present but no disulphates. Only 3-sulphates were found. Both sulphates and glucuronides were found exclusively as glycine or taurine conjugates and no such derivatives of unconjugated bile acids were isolated.

The isolated conjugates were split either by a combination of acid solvolysis and alkaline hydrolysis or by Helix pomatia and cholylglycine hydrolase.

Key Words:

• capillary column
• gas chromatography
• glucuronides
• ion exchange chromatography
• mass spectrometry
• reversed phase chromatography
• sulphate esters
• thin-layer chromatography