Determination of hemoglobin adducts from aldehydes formed during lipid peroxidation in vitro

Abstract

This study was performed in order to investigate the formation of hemoglobin (Hb) adducts from some aldehydes, known to be formed during the peroxidation of polyunsaturated fatty acids. Hb was reacted in vitro with aldehydes followed by reduction of hemolysate with sodium borohydride and isolation of globin. Using the N-alkyl Edman method, globin samples were derivatized with pentafluorophenyl isothiocyanate for specific splitting off of N-substituted N-terminal valines. Adducts formed were then characterized by gas chromatography mass spectrometry using different ionization techniques. It was shown that adducts from saturated aldehydes were Schiff bases and those from α,β-unsaturated aldehydes were mixtures of Schiff bases and 1,4-addition products. Aldehyde adducts were also measured in Hb from erythrocytes induced for lipid peroxidation. Incubation of the erythrocytes in presence of arachidonic acid led to increased levels of adducts.