A simple and accurate HPLC method for fecal bile acid profile in healthy and cirrhotic subjects: validation by GC-MS and LC-MS
A simple and accurate HPLC method for fecal bile acid profile in healthy and cirrhotic subjects: validation by GC-MS and LC-MS
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DOI：
10.1194/jlr.d047506
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发表时间：
2014-05-01
影响因子：
6.5
通讯作者：
Bajaj, Jasmohan S.
中科院分区：
文献类型：
--
作者：
Kakiyama, Genta;Muto, Akina;Bajaj, Jasmohan S.
我们开发了一种简单准确的高效液相色谱法，利用非共轭胆汁酸的苯那基衍生物测量粪便胆汁酸，并将其应用于肝硬化患者粪便胆汁酸的测量。HPLC法有以下步骤：1)粪便样品冻干；2)缓冲液重构和巯基甘氨酸水解酶/硫酸酯酶解偶联；3)用0.1 N NaOH在50%异丙醇中60℃孵育，水解酯化胆汁酸；4)用0.1 N NaOH从颗粒物料中提取胆汁酸；5)固相萃取法分离去共轭胆汁酸；6)用苯那基溴衍生生成苯那基酯；7)高效液相色谱分离，测定254 nm洗脱峰。HPLC测定结果与LC-MS/MS、GC-MS测定结果一致，验证了方法的有效性。然后，我们应用该方法测量了38例肝硬化患者（17例早期，21例晚期）和10例健康受试者的粪便总胆汁酸（浓度）和胆汁酸谱。晚期肝硬化患者胆汁酸浓度显著降低，提示胆汁酸合成受损。
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 degrees 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.