Reactive carbonyl species (RCS) generated by lipo- and glyco-oxidation processes, are reactive compounds that act as key factors in the development /progression of a variety of chronic diseases. Among the different approaches to prevent or restrain the RCS-induced damage, that based on a direct trapping of RCS seems to be the most promising, and represents a new therapeutic target. In this context, high reactivity and specificity towards toxic carbonyls (preservation of physiological aldehydes) and characterization of reaction products are essential in the development of new compounds. The ESI-MS approach here proposed and never previously described, allows to simultaneously obtain these information. The method involves the following steps: a) incubation of the target RCS with different QC at a 1:1 molar ratio for 3 and 24 h at 37°C; b) sample spiking with Tyr-Hys as internal standard (final concentration 0.4 mM) and 1:4 dilution with H2O:CH3CN (70:30 v/v) for infusion experiments (triple quadrupole Quantum Ultra, equipped with ESI and APCI sources). The quenching efficiency (reactivity) is determined by monitoring the QC consumption in respect to the internal standard (ion response of the respective protonated or deprotonated molecular ions). When QC consumption is over 10%, reaction products are searched and characterized by MS2. The method has been validated using well-known QC such as GSH, carnosine, pyridoxamine, aminoguanidine and D-penicillamine, and the most reactive and cytotoxic target RCS: 4-hydroxy-trans-2-nonenal (HNE), glyoxal, methylglyoxal, 15-deoxy-12,14-Prostaglanidin J2. Specificity is determined using pyridoxalphosphate (PP) as target physiological aldehyde. The results allowed to obtain the order of reactivity of the different QC toward the RCS, which in the case of HNE, was GSH> carnosine>aminoguanidine>pyridoxamine. At the same time, it was possible to identify the corresponding reaction products: Michael adducts for GSH and carnosine, Schiff base adducts for aminoguanidine and pyridoxamine. Other adducts (3-amino-1,2,4-triazine derivatives) were identified as reaction products of aminoguanidine and dialdehydes. Aminoguanidine was confirmed to cross-react with unreactive physyiological aldehydes (PP), with formation of the corresponding Schiff base. These results, in accordance to literature data obtained using multiple analytical strategies, underline the usefulness of this approach that permits to obtain a set of information within a single MS analysis.

ESI-MS infusion in the development of carbonyl quenchers : a rapid and versatile tool to study reactivity, specificity and reaction mechanisms / G. Aldini, L. Gamberoni, M. Orioli, L. Regazzoni, R. Maffei Facino, M. Carini. ((Intervento presentato al 24. convegno Informal Meeting on Mass Spectrometry tenutosi a Ustron nel 2006.

ESI-MS infusion in the development of carbonyl quenchers : a rapid and versatile tool to study reactivity, specificity and reaction mechanisms

G. Aldini
Primo
;
L. Gamberoni
Secondo
;
M. Orioli;L. Regazzoni;R. Maffei Facino
Penultimo
;
M. Carini
Ultimo
2006

Abstract

Reactive carbonyl species (RCS) generated by lipo- and glyco-oxidation processes, are reactive compounds that act as key factors in the development /progression of a variety of chronic diseases. Among the different approaches to prevent or restrain the RCS-induced damage, that based on a direct trapping of RCS seems to be the most promising, and represents a new therapeutic target. In this context, high reactivity and specificity towards toxic carbonyls (preservation of physiological aldehydes) and characterization of reaction products are essential in the development of new compounds. The ESI-MS approach here proposed and never previously described, allows to simultaneously obtain these information. The method involves the following steps: a) incubation of the target RCS with different QC at a 1:1 molar ratio for 3 and 24 h at 37°C; b) sample spiking with Tyr-Hys as internal standard (final concentration 0.4 mM) and 1:4 dilution with H2O:CH3CN (70:30 v/v) for infusion experiments (triple quadrupole Quantum Ultra, equipped with ESI and APCI sources). The quenching efficiency (reactivity) is determined by monitoring the QC consumption in respect to the internal standard (ion response of the respective protonated or deprotonated molecular ions). When QC consumption is over 10%, reaction products are searched and characterized by MS2. The method has been validated using well-known QC such as GSH, carnosine, pyridoxamine, aminoguanidine and D-penicillamine, and the most reactive and cytotoxic target RCS: 4-hydroxy-trans-2-nonenal (HNE), glyoxal, methylglyoxal, 15-deoxy-12,14-Prostaglanidin J2. Specificity is determined using pyridoxalphosphate (PP) as target physiological aldehyde. The results allowed to obtain the order of reactivity of the different QC toward the RCS, which in the case of HNE, was GSH> carnosine>aminoguanidine>pyridoxamine. At the same time, it was possible to identify the corresponding reaction products: Michael adducts for GSH and carnosine, Schiff base adducts for aminoguanidine and pyridoxamine. Other adducts (3-amino-1,2,4-triazine derivatives) were identified as reaction products of aminoguanidine and dialdehydes. Aminoguanidine was confirmed to cross-react with unreactive physyiological aldehydes (PP), with formation of the corresponding Schiff base. These results, in accordance to literature data obtained using multiple analytical strategies, underline the usefulness of this approach that permits to obtain a set of information within a single MS analysis.
Settore CHIM/08 - Chimica Farmaceutica
ESI-MS infusion in the development of carbonyl quenchers : a rapid and versatile tool to study reactivity, specificity and reaction mechanisms / G. Aldini, L. Gamberoni, M. Orioli, L. Regazzoni, R. Maffei Facino, M. Carini. ((Intervento presentato al 24. convegno Informal Meeting on Mass Spectrometry tenutosi a Ustron nel 2006.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/152050
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