Many studies have provided direct evidence that reactive oxygen species (ROS) generated in skeletal muscles during contraction, initiate a cascade of events that lead to impairment of functional proteins (i.e. actin). In detail, the intermediate reactive carbonyl species (RCS), generated by lipid peroxidation of polyunsaturated fatty acids (PUFAs), play a key role, through a covalent modification of nucleophilic sites on proteins (Michael type adduction to Cys, His and Lys residues). Detoxification of the most reactive RCS, ,-unsaturated aldehydes such as 4-hydroxy-trans-2-nonenal (HNE) and acrolein (ACR), has been extensively studied in several biological systems such as erythrocytes and liver slices and mainly involves conjugation with GSH to give Michael adducts. No data exist on the fate of these compounds in skeletal muscle, highly susceptible to peroxidative attack. In this study, through a peptidomic approach carried out by LC-ESI-MS/MS, and spiking skeletal muscle with HNE, we found that besides GSH, endogenous histidine-containing di-peptides such as carnosine (-alanyl-L-histidine, CAR) and anserine (-alanyl-L-1-methylhistidine, ANS) are involved in aldehydes detoxification. The carbonyl trapping ability of CAR and ANS has been further investigated in vitro by monitoring HNE/ACR consumption, and by elucidating the reaction products and the mechanisms of reaction by a combined NMR and ESI-MS/MS. CAR reacts with HNE through a mechanism involving the amino group of -alanine, (formation of a imine intermediate), and the N atom of the imidazole ring of the L-His residue which forms a covalent bond with C3 of HNE, leading to a Micahel adduct. Modeling studies (molecular dynamics simulations in water) on the imine intermediate confirm the reaction mechanism, evidencing that the imine intermediate preferentially assumes conformers in which the imidazole ring approaches the C3 of HNE moiety. Finally, to confirm the detoxifying role of these nucleophilic peptides, an innovative LC-MS/MS method was developed and validated to determine HNE conjugated adducts with GSH and histidine-containing dipeptides in rat skeletal muscle homogenates exposed to a flux of peroxyl radicals generated by the radical initiator AAPH.

A LC-MS /MS peptidomic approach for the functional screening of endogenous carbonyl quenchers in biological matrices / G. Aldini, M. Orioli, G. Vistoli, G. Beretta, L. Gamberoni, M. Carini. ((Intervento presentato al 11. convegno Meeting on Recent Development in Pharmaceutical Analysis (RDPA) tenutosi a Rimini nel 2005.

A LC-MS /MS peptidomic approach for the functional screening of endogenous carbonyl quenchers in biological matrices

G. Aldini
Primo
;
M. Orioli
Secondo
;
G. Vistoli;G. Beretta;L. Gamberoni
Penultimo
;
M. Carini
Ultimo
2005

Abstract

Many studies have provided direct evidence that reactive oxygen species (ROS) generated in skeletal muscles during contraction, initiate a cascade of events that lead to impairment of functional proteins (i.e. actin). In detail, the intermediate reactive carbonyl species (RCS), generated by lipid peroxidation of polyunsaturated fatty acids (PUFAs), play a key role, through a covalent modification of nucleophilic sites on proteins (Michael type adduction to Cys, His and Lys residues). Detoxification of the most reactive RCS, ,-unsaturated aldehydes such as 4-hydroxy-trans-2-nonenal (HNE) and acrolein (ACR), has been extensively studied in several biological systems such as erythrocytes and liver slices and mainly involves conjugation with GSH to give Michael adducts. No data exist on the fate of these compounds in skeletal muscle, highly susceptible to peroxidative attack. In this study, through a peptidomic approach carried out by LC-ESI-MS/MS, and spiking skeletal muscle with HNE, we found that besides GSH, endogenous histidine-containing di-peptides such as carnosine (-alanyl-L-histidine, CAR) and anserine (-alanyl-L-1-methylhistidine, ANS) are involved in aldehydes detoxification. The carbonyl trapping ability of CAR and ANS has been further investigated in vitro by monitoring HNE/ACR consumption, and by elucidating the reaction products and the mechanisms of reaction by a combined NMR and ESI-MS/MS. CAR reacts with HNE through a mechanism involving the amino group of -alanine, (formation of a imine intermediate), and the N atom of the imidazole ring of the L-His residue which forms a covalent bond with C3 of HNE, leading to a Micahel adduct. Modeling studies (molecular dynamics simulations in water) on the imine intermediate confirm the reaction mechanism, evidencing that the imine intermediate preferentially assumes conformers in which the imidazole ring approaches the C3 of HNE moiety. Finally, to confirm the detoxifying role of these nucleophilic peptides, an innovative LC-MS/MS method was developed and validated to determine HNE conjugated adducts with GSH and histidine-containing dipeptides in rat skeletal muscle homogenates exposed to a flux of peroxyl radicals generated by the radical initiator AAPH.
Settore CHIM/08 - Chimica Farmaceutica
A LC-MS /MS peptidomic approach for the functional screening of endogenous carbonyl quenchers in biological matrices / G. Aldini, M. Orioli, G. Vistoli, G. Beretta, L. Gamberoni, M. Carini. ((Intervento presentato al 11. convegno Meeting on Recent Development in Pharmaceutical Analysis (RDPA) tenutosi a Rimini nel 2005.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/145945
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