Characterization of Mercapturic Acids by Three Different Mass Spectrometric Methods

Abstract

The determination of mercapturic acids, as metabolic end-products of an important pathway in the biotransformation of xenobiotics, in human urine is becoming a standard tool for the biomonitoring of populations exposed to potential mutagens and carcinogens. Mass spectrometric methods permit the direct identification and characterization of these substances without a previous derivatization step. In this work, N-acetylcysteine conjugates of synthetic origin were characterized by three different mass spectrometric methods: matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, positive and negative californium-252 plasma desorption mass spectrometry (PD-MS) and fast-atom bombardment (FAB) mass spectrometry. For PD-MS, samples were prepared by the spin deposition technique on plain aluminium targets. α-Cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid were used as MALDI matrices and glycerol as a matrix for FAB. Complementary structural information on the N-acetylcysteine moiety and the xenobiotic moiety was obtained through PD-MS and FAB spectra. All three techniques could be used for relative molecular mass determination. With the ever increasing awareness and knowledge about the potential toxicity of environmental and industrial chemicals to humans, animals and other living organisms, there is an increasing need for reliable methods for the biomonitoring of populations potentially exposed to occupational or environmental pollutants. In this regard, much attention has been paid to biomonitoring of exposure to potential mutagens and carcinogens.1,2 Many compounds which are carcinogens or mutagens, as such or after biotransformation to mutagenic or carcinogenic metabolites, are electrophilic in nature and, as a result, they react with nucleophilic sites in essential macromolecular proteins or nucleic acids.3 Alternatively, methods for biomonitoring of exposure to these chemicals rely on the determination of the parent chemical or its metabolites in biological fluids, such as blood or urine. Mercapturic acids, S-substituted-N-acetyl-L-cysteinederivatives, are metabolic end-products of an important pathway in the biotransformation of xenobiotics, notably the glutathione conjugation pathway. Substrates possessing an electrophilic centre, such as alkyl or aromatic halides, react with an endogenous nucleophile, such as tripeptide glutathione ( -Glu-Cys-Gly; GSH).5 This reaction is usually catalysed by cytosolic or microsomal GSH transferases present in cells of the liver, blood and to a lesser extent other organs. However, it can also occur spontaneously.Once the GSH conjugates have been formed in the liver, intestine or kidney, the glutamyl moiety is removed by the action of -glutamyl transpeptidases. This is followed by removal of glycine by cysteinyl glycinase and N-acetylation of the remaining S-alkylated cysteine conjugate (Fig. 1) to give rise to the mercapturic acids. These are often the major urinary products of GSH conjugation in vivo, although cysteine conjugates and other sulphur-conta...