Duane W C, Schteingart C D, Ton-Nu H T, Hofmann A F
Department of Medicine, Veterans Affairs Medical Center, Minneapolis, MN 55417, USA.
J Lipid Res. 1996 Feb;37(2):431-6.
Bile acids labeled with 3H on the sterol nucleus lose a substantial fraction of label during enterohepatic cycling and conversion to secondary bile acids. We tested the isotopic stability of a side-chain 3H label, [22,23-3H]cholic acid in humans. The 3H-labeled compound was administered simultaneously with [24-14C]cholic acid to four healthy volunteers. Duodenal bile was collected daily for 5 days after isotope administration to determine the ratio of 3H/14C in bile acids. Urine was collected to determine loss of radioactivity by this route. Cholic acid and deoxycholic acid were isolated from biliary bile acids by thin-layer chromatography after deconjugation with cholylglycine hydrolase. The ratio of 3H/14C in cholic acid and deoxycholic acid remained constant and identical to that of the administered mixture in all subjects, indicating stability of the 3H label during enterohepatic cycling. Cumulative loss of 3H in urine averaged only 1.2% of administered dose and was identical to loss of 14C (average 1.3%) indicating little if any transfer of 3H from bile acid to body water. Deconjugation of biliary bile acids by alkaline hydrolysis resulted in 15-20% loss of 3H label, consistent with known base-catalyzed exchange of alpha-carbon protons on carboxylic acids. We conclude that [22,23-3H]cholic acid is a biologically stable, and therefore reliable, isotopic tracer of cholic acid in humans during enterohepatic cycling including conversion to deoxycholic acid, provided deconjugation is performed enzymatically. Because the 22,23-3H label can be inserted into most C24 bile acids, it appears the best way to tag 3H-labeled bile acids for metabolic studies.
在甾核上用³H标记的胆汁酸在肠肝循环及转化为次级胆汁酸的过程中会损失相当一部分标记。我们测试了人侧链³H标记物[22,23-³H]胆酸的同位素稳定性。将³H标记的化合物与[24-¹⁴C]胆酸同时给予四名健康志愿者。同位素给药后连续5天每天收集十二指肠胆汁，以测定胆汁酸中³H/¹⁴C的比率。收集尿液以确定经此途径的放射性损失。用胆酰甘氨酸水解酶进行脱共轭后，通过薄层色谱从胆汁胆汁酸中分离出胆酸和脱氧胆酸。在所有受试者中，胆酸和脱氧胆酸中³H/¹⁴C的比率保持恒定，且与给药混合物的比率相同，这表明³H标记在肠肝循环过程中具有稳定性。尿液中³H的累积损失平均仅为给药剂量的1.2%，与¹⁴C的损失（平均1.3%）相同，这表明³H从胆汁酸向体内水的转移极少（如果有的话）。碱性水解使胆汁胆汁酸脱共轭导致³H标记损失15 - 20%，这与已知的羧酸α-碳原子质子的碱催化交换一致。我们得出结论，[22,23-³H]胆酸在包括转化为脱氧胆酸的肠肝循环过程中是一种生物学稳定且因此可靠的人胆酸同位素示踪剂，前提是脱共轭是通过酶促进行的。由于22,23-³H标记可以插入大多数C24胆汁酸中，它似乎是为代谢研究标记³H标记胆汁酸的最佳方法。
