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

Genta Kakiyama,Akina Muto,Hajime Takei,Hiroshi Nittono,Tsuyoshi Murai,Takao Kurosawa,Alan F Hofmann,William M Pandak,Jasmohan S Bajaj

doi:10.1194/jlr.d047506

Abstract

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.

Highlights

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
Major modifications include deconjugation followed by dehydroxylation at C-7, by which CA is converted to deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) is converted to lithocholic acid (LCA)
0.00 a Sum of all the bile acid species examined by this method. b Sum of CA, CDCA, DCA, LCA, isoDCA, isoLCA, ursodeoxycholic acid (UDCA), and hyocholic acid (HCA) which are common bile acid species examined by three different methods

Summary

EXPERIMENTAL PROCEDURES

Abbreviations used for bile acids in this paper are based on the nomenclature recommendations reported by Hofmann et al (10) with a few exceptions: We have used semi-trivial nomenclature for the ⌬1-3-one, ⌬4-3-one, and ⌬4,6-3-one derivatives of the common bile acids by using the abbreviation for the saturated compound. Steps in the sample preparation procedures for the three different methods of fecal bile acid analysis. For the preparation of standard stock solutions, unconjugated bile acids were dissolved in 90% ethanol at a concentration of 200 ␮g/ml (500 nmol/ml). Each concentration of bile acid phenacyl ester mixture was dissolved in 200 ␮l of 90% methanol and a 20 ␮l aliquot was injected to the HPLC. For the recovery rate test, 100 ␮l aliquots of 50 or 500 nmol/ ml stock solutions were spiked into the dried stool and the samples were subjected to the above entire clean-up and derivatization process (method A, without alkaline hydrolysis). Analysis of fecal bile acids by GC-MS Lyophilized stool powder (5.0 mg, weighed exactly) was heated with water, and incubated with cholylglycine hydrolase/sulfatase for 16 h as described in the HPLC section. The mass spectra were recorded at an ionization energy of 70 eV with an ion source temperature of 250°C

RESULTS

DISCUSSION

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