A simple and accurate HPLC method for fecal bile acid profile in healthy and cirrhotic subjects: validation by GC-MS and LC-MS[S]

• Published in: Journal of lipid research Vol. 55; no. 5; pp. 978 - 990
• Main Authors: Kakiyama, Genta, Muto, Akina, Takei, Hajime, Nittono, Hiroshi, Murai, Tsuyoshi, Kurosawa, Takao, Hofmann, Alan F., Pandak, William M., Bajaj, Jasmohan S.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.05.2014; The American Society for Biochemistry and Molecular Biology; Elsevier
• Subjects: bile acid 24-phenacyl ester; Bile Acids and Salts - analysis; Bile Acids and Salts - chemistry; Case-Control Studies; Chromatography, High Pressure Liquid - methods; derivatization; esterified bile acids; extraction; Feces - chemistry; Female; Fibrosis; Gas Chromatography-Mass Spectrometry; high-performance liquid chromatography; Humans; liquid chromatography-tandem mass spectrometry; liver cirrhosis; Male; Methods; Middle Aged; routine analysis; bile acid 24-phenacyl ester; gas chromatography-mass spectrometry; esterified bile acids; routine analysis; liquid chromatography-tandem mass spectrometry; high-performance liquid chromatography; derivatization; extraction; liver cirrhosis
• ISSN: 0022-2275; 1539-7262; 1539-7262
• DOI: 10.1194/jlr.D047506

We have developed a simple and accurate HPLC method for measurement of fecal bile acids using phenacyl derivatives of unconjugated bile acids, and applied it to the measurement of fecal bile acids in cirrhotic patients. The HPLC method has the following steps: 1) lyophilization of the stool sample; 2) reconstitution in buffer and enzymatic deconjugation using cholylglycine hydrolase/sulfatase; 3) incubation with 0.1 N NaOH in 50% isopropanol at 60°C to hydrolyze esterified bile acids; 4) extraction of bile acids from particulate material using 0.1 N NaOH; 5) isolation of deconjugated bile acids by solid phase extraction; 6) formation of phenacyl esters by derivatization using phenacyl bromide; and 7) HPLC separation measuring eluted peaks at 254 nm. The method was validated by showing that results obtained by HPLC agreed with those obtained by LC-MS/MS and GC-MS. We then applied the method to measuring total fecal bile acid (concentration) and bile acid profile in samples from 38 patients with cirrhosis (17 early, 21 advanced) and 10 healthy subjects. Bile acid concentrations were significantly lower in patients with advanced cirrhosis, suggesting impaired bile acid synthesis.