Identification of Novel Metabolites of Colchicine in Rat Bile Facilitated by Enhanced Online Radiometric Detection

Abstract

ABSTRACT:Three novel conjugation metabolites of colchicine were identified in rat bile facilitated by enhanced on-line liquid chromatographyaccurate radioisotope counting. The known 2-and 3-demethylcolchicines (DMCs) underwent O-sulfate conjugation in addition to the previously described O-glucuronidation. 2-DMC was preferably O-glucuronidated, whereas 3-DMC predominantly yielded O-sulfation conjugates, indicating phase II conjugation regiopreferences. Moreover, M1 was identified as a novel glutathione conjugate and a possible biotransformation pathway for its formation was proposed. The known 2-DMC (M6), 3-DMC (M7), 2-DMC glucuronide (M4), and novel 3-DMC sulfate (M3) were confirmed as the major metabolites. Radiometric data were acquired by the XFlow liquid chromatography-accurate radioisotope counting (XFlow LC-ARC) system, a novel technology for dynamic control of both on-column and postcolumn high-performance liquid chromatography flow rates to maximize sensitivity and resolution of radiochromatograms. A comparative evaluation was also performed between the XFlow LC-ARC system and a conventional flow radiometric detection system using bile samples from an in vivo disposition study of colchicine in male Sprague-Dawley rats. Results demonstrated a 20-fold sensitivity improvement of the XFlow LC-ARC system in comparison with radioactivity detection by conventional flow scintillation analyzers. The dynamic flow mode also provided the best chromatographic resolution. Unambiguous metabolite identification was performed by high-resolution mass spectrometry and nuclear magnetic resonance analysis.

Hepatobiliary Toxicity of Furan: Identification of Furan Metabolites in Bile of Male F344/N Rats

“…Retention times and mass spectral data of these compounds were consistent with synthesized reference compounds, i.e., a cysteinylglycine-BDA-GSH conjugate d (m/z 532), a cysteinyl-BDA-GSH conjugate e (m/z 475), and a GSH-BDA-glycine conjugate f (m/z 429). The structure of f is further supported by the characteristic neutral loss of 129, resulting from cleavage of the ␥-glutamyl-cysteinyl-isopeptide bond within a GSH residue and loss of 273 (GSH cleavage at the SCH 2 group) (Xu et al, 2008), which indicate that GSH is linked to the pyrrole ring by its thiol group and not by an amino group. The amount of metabolite f was below the limit of detection in bile samples from [3,4-13 C]furantreated rats.…”

Studies on hepatotoxicity and toxicokinetics of colchicine

“…O ‐demethylation is a major pathway for COL metabolism in which P450 3A plays a critical role in the process. O ‐ glucuronidated metabolites, O ‐sulfation conjugates, and glutathione conjugates derived from demethylated COL were detected in rat bile . In our early work, we detected several cysteine conjugates derived from demethylated COL in COL‐exposed microsomal protein samples after proteolytic digestion.…”

A New Insight into Toxicity of Colchicine Analogues by Molecular Docking Analysis Based on Intestinal Tight Junction Protein ZO-1

“…As discussed above, the planar structure of tropolone ring C and the hydrophobic methoxyl at C-10 greatly impacted COL analogues' activities by affecting their interaction with the relevant protein. Herein, a total of 13 COL analogues (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31) were involved in molecular docking analysis to understand better how the modification on ring C would affect ZO-1-related intestinal injury toxicity of COL. Chemically, these ligands with a type II structure, as shown in Figure 1, were all obtained from chemical modification at C-10.…”