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Journal of Lipid Research, Vol. 18, 339-362, May 1977
Copyright © 1977 by Lipid Research, Inc.

Analysis of metabolic profiles of bile acids in urine using a lipophilic anion exchanger and computerized gas-liquid chromatography-mass spectrometry

B. Almé , A. Bremmelgaard , J. Sjövall , and P. Thomassen

Department of Chemistry, Karolinska Institutet, S-104 01 Stockholm 60, Sweden

A method is described for quantitative analysis of bile acids in urine. Urine is acidified and bile acids are extracted on an Amberlite XAD-2 column. Bile salts are converted to acids on an Amberlyst A-15 column and are separated into groups of unconjugated, glycine, taurine, monosulfated, and polysulfated conjugates using the lipophilic anion exchanger diethylaminohydroxypropyl Sephadex LH-20 (DEAP-LH-20). After solvolysis and hydrolysis, the deconjugated bile acids are purified on DEAP-LH-20, and are converted to methyl ester trimethylsilyl ether derivatives. Identification and quantitation of the individual bile acids is accomplished by computerized gas-liquid chromatography-mass spectrometry.

The daily excretion of bile acids in urine from healthy subjects was 6.4-11 µmoles. The mixture of bile acids was quite complex and differed from that in bile. About 30 bile acids were identified or partially characterized. Three of these were monosubstituted: lithocholic, allolithocholic, and 3ßbeta;-hydroxy-5-cholenoic acids. Fourteen disubstituted bile acids included epimers of deoxycholic, allodeoxycholic, chenodeoxycholic, allochenodeoxycholic, and hyodeoxycholic acids. 3-Hydroxy-12-keto-5ßbeta;-cholanoic acid was the major ketonic bile acid and 3ßbeta;,12-dihydroxy-5-cholenoic acid was the major unsaturated bile acid in this group. Nine trihydroxy bile acids included cholic and allocholic acids, epimers of these compounds, hyocholic acid, and a 1-hydroxylated bile acid tentatively characterized as 1,3,12-trihydroxycholanoic acid. Cholestatic subjects excreted tetrahydroxycholanoates carrying hydroxyl groups in positions 1, 3, 6, 7, 12, or 23.

All monohydroxy and the predominant part of dihydroxy bile acids were present in the monosulfate fraction. Exceptions were 3,12ßbeta;-dihydroxy- and 3-hydroxy-12-keto-5ßbeta;-cholanoic acids, which were found mainly in the glycine conjugate fraction. Most of the trihydroxy bile acids were nonsulfated, and cholic and norcholic acids were the major unconjugated bile acids. The tetrahydroxy bile acids and hyocholic acid were present mainly in the taurine conjugate fraction, while 1,3,12-trihydroxycholanoic acid was predominantly found in the glycine conjugate fraction. Sulfation of trihydroxy bile acids was increased in patients with marked cholestasis. All bile acids in the monosulfate fraction were conjugated and carried the sulfate ester group at C-3. Significant amounts of di- and trisulfates were not found.

The results indicate selective mechanisms for sulfation, hydroxylation, and renal elimination of bile acid conjugates. Analysis of metabolic profiles of bile acids in urine may be a useful method in studies of the function of organs involved in bile acid metabolism.

Supplementary key words ion exchange chromatography • lipophilic Sephadex • Lipidex

Submitted on July 2, 1976
Accepted on December 2, 1976

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