Immunochemical Detection of a Fluorophore Derived from the Lipid Peroxidation Product 4-Hydroxy-2-Nonenal and Lysine

Abstract

Aging and the progression of certain degenerative diseases are accompanied by increases in intra- and intercellular fluorescent material, termed lipofuscin and ceroid, respectively (for review, see refs. 1,2. These pigments are observed within granules composed, in part, of damaged protein and lipid. Modification of various biomolecules by aldehyde products of lipid peroxidation is believed to contribute to lipofuscin and ceroid formation. However direct evidence demonstrating the occurrence of lipid peroxidation product-derived fluorophores on protein components of lipofuscin or ceroid has yet to be obtained (for review, see refs. 1,2). We have recently identified a fluorescent product formed in the reaction of N_α-acetyllsyine (NAL) and 4-hydroxy-2-nonenal (HNE) (3), a major product of lipid peroxidation and the most reactive of these compounds under physiological conditions (4). This fluorescent compound, characterized as a2-hydroxy-3-imino-1,2-dihydropyrrol derivative (3, R₁ = R₂ = NAL), appears to form upon oxidative cyclization of the nonfluorescent 2:1 lysine-HNE Michael adduct-Schiff base cross-link (Scheme 1). Polyclonal antibody (PAb) to the NAL-HNE fluorophore was raised in rabbit and found to be highly specific to the chromophore structure of the compound (3). This antibody has been used to demonstrate the formation of the lysineHNE derivative of this fluorophore upon exposure of protein to HNE in vitro.