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-trihydrox y-5 beta-cholanoic acid (cholic acid) with adenosine 5'-monophosphate havin g an acid anhydride linkage, has been characterized by means of liquid chro matography/mass spectrometry in an incubation mixture with a rat liver micr osomal fraction. The authentic specimen of cholyl-adenylate was synthesized using the carbodiimide method and the structure was confirmed by MS and nu clear 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-ad enylate and a CoA thioester of cholic acid (cholyl-CoA) were monitored with characteristic negative ions of m/z 736 and 577, respectively. Cholyladeny late was definitely characterized and preferential biotransformation into t he acyl-adenylate prior to formation of cholyl-CoA was noted. The nonenzyma tic formation of taurine-conjugated cholic acid by incubation of cholyl-ade nylate with taurine in a buffer solution was also demonstrated. (C) 1999 Ac ademic Press.