Polyclonal Antibodies to a Fluorescent 4-Hydroxy-2-nonenal (HNE)-Derived Lysine−Lysine Cross-Link: Characterization and Application to HNE-Treated Protein and in Vitro Oxidized Low-Density Lipoprotein

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Abstract
Oxidative modification of low-density lipoprotein (LDL) is thought to play a key role in the etiology of atherosclerosis. Oxidized LDL that accumulates in atherosclerotic plaques is known to exhibit a characteristic fluorescence with excitation and emission near 360 and 430 nm, respectively. (E)-4-Hydroxy-2-nonenal (HNE), formed during LDL oxidation, is capable of modifying LDL to generate the same fluorescent signature. The HNE-derived fluorophore was shown by us to possess a 2-hydroxy-2-pentyl-1,2-dihydropyrrol-3-one iminium (HPDPI) structure. We herein report the synthesis of the HPDPI-derived lysine−lysine cross-link needed as a standard reference for HPLC quantitation of the cross-link in protein hydrolysates. The main focus of the current work, however, is the design and development of two polyclonal antibodies against the HPDPI epitope. Utilizing these antibodies, levels of the HPDPI epitope were estimated in HNE-treated bovine serum albumin and in copper-oxidized LDL by an enzyme-linked immunosorbent assay. Our results are consistent with the premise that the fluorescent HPDPI cross-link is a key contributor to the fluorescence exhibited by atherosclerotic lesions.