Immunochemical Evidence Supporting 2-Pentylpyrrole Formation on Proteins Exposed to 4-Hydroxy-2-nonenal

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Abstract
Previous model studies suggested the formation of lysine-based 2-pentylpyrroles as novel late adduction products formed upon exposure of proteins to the lipid peroxidation product 4-hydroxy-2-nonenal (HNE). Two 2-pentylpyrrole immunogens were prepared, one by treating keyhole limpet hemocyanin (KLH) directly with 4-oxononanal and the other by preformation of 6-(2-pentylpyrrol-1-yl)hexanoic acid from 6-aminocaproic acid and 4-oxononanal, followed by carbodiimide coupling to KLH. Pyrrole content and lysine modification in KLH were assayed independently. Following immunization of rabbits, antibody titer increased and plateaued over a 4 month period. The structural specificity of the IgG fractions of the antisera was evaluated through comprehensive competitive ELISA studies. These antibodies were used to verify the time-dependent appearance of the 2-pentylpyrrole epitope in protein exposed to HNE. Potential advantages of antibodies recognizing “advanced lipid peroxidation end products” over those recognizing "early" HNE adduction products are discussed.