The steps used in identification of metabolites were (a) solvolysis,(b) hydrolysis, (c) extraction, (d) methylation and thin-layer chromatography,(e) column chromatography using Glycophase G onControlled-Pore Glass 80-100 mesh (Pierce Chemical Co.), (f ) HPLCusing FPorasil (lo-@ silica, Waters Associates) and reverse isotopedilution. All these steps have been described in detail in previouspublications (8, 11)including the quantitation of radioactivity byliquid scintillation spectrometry using a Beckman CPM 200 instrument.
In dual isotope studies, narrow windows were used so that lessthan 1% of the 3H was detected in the “C channel and crossover of14C into the tritium window was 10%. The amount of each metabolitein bile was determined from the proportional distribution of radioactivityafter thin-layer chromatography and confirmed by HPLC
using PPorasil as described above. It was found that complete separationof lithocholic acid from 3@-hydroxy-5-cholenoic acid and of
chenodeoxycholic acid from deoxycholic acid (3cu,l2a-dihydroxy-5@-cholanoic acid) could be obtained using 20 X 20-cm plates coatedwith Silica Gel G and a solvent system of chloroform:acetone (9~1,v/v) followed after drying at room temperature byisooctane :isopropylether:glacial acetic acid (2:2:1, v/v).
Radioactive lithocholic, deoxycholic, and cholic acids were purchasedfrom Amersham/Searle and/or New England Nuclear. Radioactive3-keto-5&cholanoate and 3cu-hydroxy-5@-cholanoate were prepared from lithocholic acid by chromate oxidation and sodium borohydridereduction.
Chenodeoxycholic Acid compounds were purified by thin-layer andcolumn chromatography and used as standards.
For intravenous administration of tracer amounts of radioactivebile acids, the compounds were dissolved in 0.05 ml of absoluteethanol and added with vortexing to 5 ml of sterile 5% dextrose in0.9%
Chenodeoxycholic Acid containing 5% human serum albumin. Studies in hamstersrequired larger amounts of bile acids; and for this purpose, the ‘H/
“C isotope ratio of lithocholic and 3B-hydroxy-5-cholenoic acids wasadjusted to provide an approximate $1 counting ratio. Then, nonradioactive bile acids were added to provide a final total concentration
of 900 nmol/ml. This mixture was prepared initially in methanol andthen taken to dryness using vacuum. The compounds were thenredissolved with heating to 60 “C in 0.2 ml of propylene glycol towhich 0.8 ml of the sterile solution containing albumin as describedabove was added.
Chenodeoxycholic Acid that required T-tube drainage following cholecystectomyfor gallstones consented to the intravenous administration
of tracer amounts of bile acids.
Male and female New Zealand rabbits weighing between 1.5 and 4kg received intravenous pentobarbital anesthesia, which was maintainedthroughout the study. The abdomen was opened by a midlineincision, and a PE 90 polyethylene cannula was inserted into thecommon duct and brought out through the incision, which was thenclosed. Bile was drained into graduated tubes for the determinationof volume.
