Quantitative Analysis of Acrolein-Specific Adducts Generated during Lipid Peroxidation–Modification of Proteins in Vitro: Identification of Nτ-(3-Propanal)histidine as the Major Adduct

Abstract

Acrolein, a ubiquitous pollutant in the environment, is endogenously formed through oxidation reactions and is believed to be involved in cytopathological effects observed during oxidative stress. Acrolein exerts these effects because of its facile reactivity with biological materials, particularly proteins. In the present study, we quantitatively analyzed the acrolein-specific adducts generated during lipid peroxidation-modification of proteins and identified the acrolein adduct most abundantly generated in the in vitro oxidized low-density lipoproteins (LDL). Taking advantage of the fact that the acrolein-lysine adducts, N(ε)-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine) and N(ε)-(3-methylpyridinium)lysine (MP-lysine), have stable core structures resistant to the acid hydrolysis condition of proteins, we examined the formation of these adducts in proteins using high performance liquid chromatography with online electrospray ionization tandem mass spectrometry. However, only MP-lysine was detected as a minor product in the iron/ascorbate-mediated oxidation of polyunsaturated fatty acids in the presence of proteins and in the oxidized low-density lipoproteins (LDL). However, using a reductive amination-based pyridylamination method, we analyzed the acrolein-specific adducts with a carbonyl functionality and found that acrolein modification of the protein produced a number of carbonylated amino acids, including an acrolein-histidine adduct. On the basis of the chemical and spectroscopic evidence, this adduct was identified as N(τ)-(3-propanal)histidine. More notably, N(τ)-(3-propanal)histidine appeared to be one of the major adducts generated in the oxidized LDL. These data suggest that acrolein generated during lipid peroxidation may primarily react with histidine residues of proteins to form N(τ)-(3-propanal)histidine.