ABSTRACT
N-Acetylcysteine (NAC) conjugates of the five major bile acids occurring in man were synthesized in order to investigate the possible formation in vivo of these conjugates. Upon collision-induced dissociation, structurally informative daughter ions were observed. The transformation of cholyl-adenylate and cholyl-CoA thioester into a N-acetyl-S-(cholyl)cysteine by rat hepatic glutathione S-transferase was confirmed by liquid chromatography/electrospray ionization-linear ion trap mass spectrometry (LC/ESI-MS(2)). Lithocholic acid was administered orally to bile duct-ligated rats that also received NAC intraperitoneally. The NAC conjugate of lithocholic acid was identified in urine by means of LC/ESI-MS(2). Rapid hydrolysis of the BA-NAC conjugates by rabbit liver carboxylesterase was found, demonstrating the possible labile nature of the NAC conjugates formed in the liver.
Subject(s)
Acetylcysteine/metabolism , Bile Acids and Salts/metabolism , Cholestasis/metabolism , Chromatography, Liquid/methods , Spectrometry, Mass, Electrospray Ionization/methods , Acetylcysteine/chemical synthesis , Acetylcysteine/chemistry , Animals , Bile Acids and Salts/chemical synthesis , Bile Acids and Salts/chemistry , Carboxylesterase/metabolism , Disease Models, Animal , Humans , Liver/chemistry , Liver/metabolism , Male , Rabbits , RatsABSTRACT
Acyl-adenylates and acyl-CoA thioesters of bile acids (BAs) are reactive acyl-linked metabolites that have been shown to undergo transacylation-type reactions with the thiol group of glutathione (GSH), leading to the formation of thioester-linked GSH conjugates. In the current study, we examined the transformation of cholyl-adenylate (CA-AMP) and cholyl-coenzyme A thioester (CA-CoA) into a cholyl-S-acyl GSH (CA-GSH) conjugate by rat hepatic glutathione S-transferase (GST). The reaction product was analyzed by liquid chromatography (LC)/electrospray ionization (ESI)-linear ion trap mass spectrometry (MS). The GST-catalyzed formation of CA-GSH occurred with both CA-AMP and CA-CoA. Ursodeoxycholic acid, lithocholic acid, and 2,2,4,4-(2)H4-labeled lithocholic acid were administered orally to biliary fistula rats, and their corresponding GSH conjugates were identified in bile by LC/ESI-MS2. These in vitro and in vivo studies confirm a new mode of BA conjugation in which BAs are transformed into their GSH conjugates via their acyl-linked intermediary metabolites by the catalytic action of GST in the liver, and the GSH conjugates are then excreted into the bile.
Subject(s)
Bile Acids and Salts/chemistry , Bile/chemistry , Glutathione/chemistry , Acyl Coenzyme A/chemistry , Acyl Coenzyme A/metabolism , Animals , Bile Acids and Salts/analysis , Bile Acids and Salts/metabolism , Catalysis , Chromatography, Liquid , Glutathione/analysis , Glutathione/metabolism , Male , Rats , Rats, Wistar , Spectrometry, Mass, Electrospray IonizationABSTRACT
The formation of thioester-linked glutathione (GSH) conjugates of bile acids (BAs) is presumed to occur via trans-acylation reactions between GSH and reactive acyl-linked metabolites of BAs. The present study examines the chemical reactivity of cholyl-adenylate and cholyl-CoA thioester, acyl-linked metabolites of cholic acid (CA), with GSH to form CA-GSH conjugate in vitro. The authentic specimen of CA-GSH was synthesized along with GSH conjugates of four common BAs found in the human body. Their structures were confirmed by proton-nuclear magnetic resonance spectroscopy and electrospray ionization (ESI)-tandem mass spectrometry in positive- and negative-ion modes. Incubation of cholyl-adenylate or cholyl-CoA thioester with GSH was carried out at pH 7.5 and 37 degrees C for 30 min, with analysis of the reaction mixture by liquid chromatography/ESI-tandem mass spectrometry, where CA-GSH was detected on the product ion mass chromatograms monitored with stable and abundant dehydrated positive-ion [M+HH(2)O](+) at m/z 680.3 and fragmented negative-ion [GSHH](-) at m/z 306.0, and was definitely identified by CID spectra by comparison with those of the authentic sample. The results show that both cholyl-adenylate and cholyl-CoA thioester are able to acylate GSH in vitro.
