Disposition of glutathione conjugates in rats by a novel glutamic acid pathway: characterization of unique peptide conjugates by liquid chromatography/mass spectrometry and liquid chromatography/NMR

With the advent of liquid chromatography/mass spectrometry and liquid chrom atography/NMR, it has become easier to characterize metabolites that were o nce difficult to isolate and identify. These techniques have enabled us to uncover the existence of an alternate pathway for the disposition of glutat hione adducts of several structurally diverse compounds. Studies were carri ed out using acetaminophen as a model compound to investigate the role of t he glutamic acid pathway in disposition of the glutathione adducts. Althoug h the mercapturic acid pathway was the major route of degradation of the gl utathione adducts, it was found that the conjugation of the glutathione, cy steinylglycine, and cysteine adducts of acetaminophen with the gamma-carbox ylic acid of the glutamic acid was both interesting and novel. The coupling of the glutathione adduct and the products from the mercapturic acid pathw ay with the glutamic acid led to unusual peptide conjugates. The natures of these adducts were confirmed unequivocally by comparisons with synthetic s tandards. This pathway (addition of glutamic acids) led to larger peptides, in contrast to the mercapturic acid pathway, in which the glutathione addu cts are broken down to smaller molecules. The enzyme responsible for the ad dition of glutamic acid to the different elements of the mercapturic acid p athway is currently unknown. It is postulated that the gamma-carboxylic aci d is activated (perhaps by ATP) before enzymatic addition to the alpha-amin o group of cysteine or glutamate takes place. The discovery of these peptid e conjugates of acetaminophen represents a novel disposition of glutathione adducts of compounds. The formation of such conjugates may represent yet a nother pathway by which drugs could produce covalent binding via their reac tive intermediates.