Characterization of cholyl-adenylate in rat liver microsomes by liquid chromatography electrospray ionization mass spectrometry

The cholyl-adenylate, covalently bound 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholanoic acid (cholic acid) with adenosine 5'-monophosphate having an acid anhydride linkage, has been characterized by means of liquid chromatography/mass spectrometry in an incubation mixture with a rat liver microsomal fraction. The authentic specimen of cholyl-adenylate was synthesized using the carbodiimide method and the structure was confirmed by MS and nuclear magnetic resonance spectroscopy. After incubation of cholic acid with a hepatic microsomal fraction in the presence of adenosine 5'-triphosphate, bile acids were extracted and purified by solid-phase extraction on a Sep-Pak C-18 cartridge and then subjected to a LC/MS analysis, where cholyl-adenylate and a CoA thioester of cholic acid (cholyl-CoA) were monitored with characteristic negative ions of m/z 736 and 577, respectively. Cholyladenylate was definitely characterized and preferential biotransformation into the acyl-adenylate prior to formation of cholyl-CoA was noted. The nonenzymatic formation of taurine-conjugated cholic acid by incubation of cholyl-adenylate with taurine in a buffer solution was also demonstrated. (C) 1999 Academic Press.