Tsaconas C, Padieu P, Maume G, Chessebeuf M, Hussein N, Pitoizet N
Anal Biochem. 1986 Sep;157(2):300-15. doi: 10.1016/0003-2697(86)90631-7.
The derivatization of bile acids into trimethylsilyl ether isobutyl ester (IBTMS) and of neutral sterols into trimethylsilyl ether (TMS) allowed the separation on an OV-1 capillary gas chromatography column of 15 bile steroids as follows: cholesterol, 7 alpha-hydroxycholesterol, 6 beta-hydroxycholesterol, 6 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol, lithocholate, deoxycholate, 25-hydroxycholesterol, chenodeoxycholate, cholate, murocholate, hyodeoxycholate, ursodeoxycholate, hyocholate, and beta-muricholate. Fragmentation data of the coupled gas chromatographic-mass spectrometric (GC-MS) analysis of these nine bile acids as IBTMS derivatives under electron impact and chemical ionizations (methane, isobutane, and ammonia) are given. The ammonia chemical ionization appears to be the best mode for compound identification and quantitation due to fragmentations into high mass ions. The comparison of methylene units of the five sterols as TMS derivatives and of each type of methyl, TMS, or isobutyl ester of the nine bile acids as TMS ethers showed that isobutyl esterification increased dramatically the retention time of the bile acids, allowing their separation after the neutral sterols. Different methods of GC-MS analysis were applied to the study of bile steroid secretion in long-term rat liver epithelial cell lines, either serum-supplemented cell lines or serum-free cell lines, growing in serum-free medium since the primary explanation or after adaptation of serum-supplemented lines to this medium. It is demonstrated for the first time that liver epithelial cell lines maintain the metabolic pathway leading from synthesized cholesterol to dioxygenated sterols and the two normal main primary bile acids of the liver, chenodeoxycholic acid and cholic acid, up to 32-47% of the in vivo daily rate, and in addition the production of alpha-muricholic acid, the bile acid marker of murine liver.
将胆汁酸衍生化为三甲基硅烷基醚异丁酯（IBTMS），将中性固醇衍生化为三甲基硅烷基醚（TMS），使得15种胆汁类固醇能够在OV-1毛细管气相色谱柱上进行分离，具体如下：胆固醇、7α-羟基胆固醇、6β-羟基胆固醇、6α-羟基胆固醇、7β-羟基胆固醇、石胆酸盐、脱氧胆酸盐、25-羟基胆固醇、鹅去氧胆酸盐、胆酸盐、鼠胆酸盐、猪去氧胆酸盐、熊去氧胆酸盐、猪胆酸盐和β-鼠胆酸盐。给出了在电子轰击和化学电离（甲烷、异丁烷和氨）条件下，对这9种胆汁酸的IBTMS衍生物进行气相色谱-质谱联用（GC-MS）分析的碎片数据。由于能裂解为高质量离子，氨化学电离似乎是化合物鉴定和定量的最佳模式。对5种作为TMS衍生物的固醇的亚甲基单元，以及9种胆汁酸作为TMS醚的每种甲基、TMS或异丁酯进行比较，结果表明异丁酯化显著增加了胆汁酸的保留时间，使其能在中性固醇之后实现分离。不同的GC-MS分析方法被应用于长期培养的大鼠肝上皮细胞系胆汁类固醇分泌的研究，这些细胞系既有补充血清的细胞系，也有在无血清培养基中生长的无血清细胞系，无血清细胞系是自原代培养起就在无血清培养基中生长，或者是补充血清的细胞系适应这种培养基之后的细胞系。首次证明肝上皮细胞系能维持从合成的胆固醇到双加氧固醇以及肝脏两种正常主要初级胆汁酸——鹅去氧胆酸和胆酸的代谢途径，其代谢率可达体内每日代谢率的32 - 47%，此外还能产生α-鼠胆酸，这是鼠肝的胆汁酸标志物。
