Acrolein sequestering ability of the endogenous tripeptide glycyl-histidyl-lysine (GHK): characterization of conjugation products by ESI-MSn and theoretical calculations.
Acrolein (ACR) is a well-known carbonyl toxin produced by lipid peroxidation of polyunsaturated fatty acids, which is involved in several life-threatening pathologies such as Alzheimer disease, arteriosclerosis, diabetes, and nephropathy. The aim of this work was to study the quenching ability of the endogenous tripeptide glycyl-histidyl-lysine (GHK), a liver cell growth factor isolated from human plasma, towards the electrophilic aldehyde ACR and to characterize the reaction products by electrospray mass spectrometry (ESI-MS/MS infusion experiments; positive ion mode). The reaction of ACR (30 microM) with GHK (0.1, 0.25, 0.5, 1.0 mM) was followed by measuring aldehyde consumption by reverse-phase HPLC (phosphate buffer, pH 7.4); after 4h, when the aldehyde had completely disappeared; the reaction products were checked by ESI-MS/MS. Several products were detected in the GHK+ACR reaction (1:1). This indicates a complex reaction cascade involving the sequential addition of ACR (up to 3 mol) to the tripeptide GHK and, in particular, to the epsilon-amino group of the lysine residue and to the N(tau) and N(pi) of the histidine moiety. The Michael addition of two molecules of ACR to the epsilon-amino group of the lysine residue is followed by aldol condensation and dehydration to give the N-(3-formyl-3,4-dehydropiperidino) derivative. The results confirm that the ESI-MS/MS approach in a direct infusion experiment permits rapid profiling of the products of the GHK+ACR reaction. They firstly point to the potential medicinal use of GHK in the prevention of carbonyl stress-linked pathologies, and--second--help shed light on the physiological role of this histidine-containing tripeptide which is claimed to be an endogenous growth factor, but has never been shown to be an ACR quencher.
Beretta
Giangiacomo
G
Istituto di Chimica Farmaceutica e Tossicologica Pietro Pratesi, University of Milan, Faculty of Pharmacy, Via Mangiagalli 25, 20133 Milan, Italy. giangiacomo.beretta@unimi.it
Arlandini
Emanuele
E
Artali
Roberto
R
Anton
Josep M Garcia
JM
Maffei Facino
R
R
D000171:Acrolein / Q000737:chemistry*; D008433:Mathematics; D009842:Oligopeptides / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D009842:Oligopeptides; C020905:glycyl-histidyl-lysine; D000171:Acrolein