Characterization of Cholyl-Adenylate in Rat Liver Microsomes by Liquid Chromatography/Electrospray Ionization–Mass Spectrometry

• Published in: Analytical biochemistry 1999-01, Vol.266 (1), p.125-132
• Main Authors: Ikegawa, Shigeo, Ishikawa, Hiroshi, Oiwa, Hiroyuki, Nagata, Masanori, Goto, Junichi, Kozaki, Teruya, Gotowda, Masaharu, Asakawa, Naoaki
• Format: Article
• Language: English
• Subjects: Acyl Coenzyme A - chemical synthesis; Acyl Coenzyme A - metabolism; Adenosine Monophosphate - analogs & derivatives; Adenosine Monophosphate - analysis; Adenosine Monophosphate - chemical synthesis; Adenosine Monophosphate - chemistry; Adenosine Triphosphate - chemistry; amidation; Animals; bile acid; Cholic Acid - chemistry; Cholic Acids - analysis; Cholic Acids - chemical synthesis; Cholic Acids - chemistry; Cholic Acids - metabolism; cholyl-adenylate; cholyl-CoA; Chromatography, Liquid - methods; electrospray ionization; liquid chromatography/mass spectrometry; Male; Mass Spectrometry - methods; Microsomes, Liver - chemistry; Microsomes, Liver - metabolism; Rats; Rats, Wistar; Taurine - chemistry; Taurocholic Acid - chemistry
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.1998.2944

The cholyl-adenylate, covalently bound 3α, 7α, 12α-trihydroxy-5β-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 C18cartridge 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 ofm/z736 and 577, respectively. Cholyl-adenylate 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.