Detoxification of cytotoxic alpha,beta-unsaturated aldehydes by carnosine: characterization of conjugated adducts by electrospray ionization tandem mass spectrometry and detection by liquid chromatography/mass spectrometry in rat skeletal muscle

Oxidation of polyunsaturated fatty acids containing phospholipids in tissue generates lipid hydroperoxides, which are further degraded to several products, among which unsaturated aldehydes such as 4-hydroxy-trans-2-nonenal (HNE) play an important role in mediating the pathological effects of oxidative stress. While the reaction of HNE with glutathione (GSH) is a well recognized pathway of detoxification in biological systems, no data are available on HNE interactions with carnosine, a dipeptide (beta-alanyl-L-histidine) present in high concentration in skeletal muscle. The aim of this work was to study the quenching ability of carnosine towards HNE and to characterize the reaction products by electrospray ionization tandem mass spectrometry (ESI-MS/MS), using GSH as a model peptide. GSH incubation with HNE in 1 mM phosphate buffer (pH 7.4) results in the complete disappearance of HNE within 1 h owing to the formation of a Michael adduct, S-(4-hydroxynonanal-3-yl)glutathione. The reaction of HNE with carnosine was studied in different molar ratios and monitored up to 24 h by high-performance liquid chromatography (HPLC) (HNE consumption), MS/MS (infusion) and liquid chromatography mass spectrometry (LC/MS) experiments. Carnosine, although less reactive than GSH, significantly quenched HNE (48.2 +/- 0.9% HNE consumption after 1 h; carnosine:HNE molar ratio 10 : 1). Two reaction products were identified: the Michael adduct, N-(4-hydroxynonanal-3-yl)carnosine involving the imidazolic nitrogen of histidine, and the imine adduct, involving the amino group of the beta-alanine residue. Definitive structure assignment was achieved by chemical reduction with NaBH(4) and multinuclear magnetic resonance experiments. To understand whether carnosine acts as a quencher of unsaturated aldehydes in biological matrices, rat skeletal muscle homogenate was incubated with HNE and the formation of conjugated adducts was determined by LC/MS analysis. Three main products were detected and identified as Michael adducts of HNE with GSH, carnosine and anserine (the N-methylated derivative of carnosine, present in high concentrations in rat muscle). The results indicate that beside GSH, histidine-containing dipeptides could be involved in the detoxification pathway of reactive aldehydes from lipid peroxidation generated in skeletal muscle during physical endurance.