Takikawa H, Otsuka H, Beppu T, Seyama Y, Yamakawa T
J Biochem. 1982 Oct;92(4):985-98. doi: 10.1093/oxfordjournals.jbchem.a134055.
Individual non-glucuronidated . non-sulfated, glucuronidated and sulfated bile acids in serum were determined, i.e. lithocholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, and cholic acid, by mass fragmentography. Glucuronic acid conjugates of lithocholic acid, deoxycholic acid, chenodeoxycholic acid, and cholic acid were synthesized via the Koenigs-Knorr condensation reaction. Deuterium labeled deoxycholic acid, lithocholic acid glucuronide, deoxycholic acid glucuronide, and deoxycholic acid sulfate were synthesized and used as internal standards. A serum sample of 1 ml including internal standards was purified with a Sep-Pak C18 cartridge. After the enzymatic cleavage of amino acid conjugates, bile acids were separated into three fractions, free, glucuronidated, and sulfated bile acids, using piperidinohydroxypropyl Sephadex LH-20 (Goto et al. (1978) Clin. Chim. Acta 87). Glucuronidated and sulfated bile acids were deconjugated by beta-glucuronidase treatment and solvolysis. Each fraction was converted to the hexafluoroisopropyl-trifluoroacetyl derivative and quantitated by mass fragmentography. The average concentrations of individual bile acid glucuronides from healthy fasting subjects (n = 9) were as follows; lithocholic acid 0.013 microgram/ml, deoxycholic acid 0.083 microgram/ml, chenodeoxycholic acid 0.078 microgram/ml, ursodeoxycholic acid 0.013 microgram/ml, and cholic acid 0.007 microgram/ml. Bile acid glucuronides occupied 7.8% of the total bile acids.
采用质量碎片分析法测定血清中未与葡萄糖醛酸结合、未硫酸化、葡萄糖醛酸化和硫酸化的单个胆汁酸，即石胆酸、脱氧胆酸、鹅去氧胆酸、熊去氧胆酸和胆酸。石胆酸、脱氧胆酸、鹅去氧胆酸和胆酸的葡萄糖醛酸共轭物通过柯尼希斯-克诺尔缩合反应合成。合成了氘标记的脱氧胆酸、石胆酸葡萄糖醛酸苷、脱氧胆酸葡萄糖醛酸苷和脱氧胆酸硫酸盐并用作内标。用Sep-Pak C18柱纯化1ml含内标的血清样品。在氨基酸共轭物经酶解后，使用哌啶羟丙基葡聚糖凝胶LH-20（后藤等人，《临床化学学报》，1978年，第87卷）将胆汁酸分离为三个部分，即游离胆汁酸、葡萄糖醛酸化胆汁酸和硫酸化胆汁酸。葡萄糖醛酸化和硫酸化胆汁酸通过β-葡萄糖醛酸酶处理和溶剂解作用进行脱共轭。每个部分转化为六氟异丙基-三氟乙酰衍生物，并通过质量碎片分析法进行定量。健康空腹受试者（n = 9）的单个胆汁酸葡萄糖醛酸苷的平均浓度如下：石胆酸0.013微克/毫升，脱氧胆酸0.083微克/毫升，鹅去氧胆酸0.078微克/毫升，熊去氧胆酸0.013微克/毫升，胆酸0.007微克/毫升。胆汁酸葡萄糖醛酸苷占总胆汁酸的7.8%。
