The lipid peroxidation product 4-hydroxy-2-nonenal: Advances in chemistry and analysis

Abstract

4-Hydroxy-2-nonenal (HNE) is one of the most studied products of phospholipid peroxidation, owing to its reactivity and cytotoxicity. It can be formed by several radical-dependent oxidative routes involving the formation of hydroperoxides, alkoxyl radicals, epoxides, and fatty acyl cross-linking reactions. Cleavage of the oxidized fatty acyl chain results in formation of HNE from the methyl end, and 9-oxo-nonanoic acid from the carboxylate or esterified end of the chain, although many other products are also possible. HNE can be metabolized in tissues by a variety of pathways, leading to detoxification and excretion. HNE-adducts to proteins have been detected in inflammatory situations such as atherosclerotic lesions using polyclonal and monoclonal antibodies, which have also been applied in ELISAs and western blotting. However, in order to identify the proteins modified and the exact sites and nature of the modifications, mass spectrometry approaches are required. Combinations of enrichment strategies with targetted mass spectrometry routines such as neutral loss scanning are now facilitating detection of HNE-modified proteins in complex biological samples. This is important for characterizing the interactions of HNE with redox sensitive cell signalling proteins and understanding how it may modulate their activities either physiologically or in disease.

Keywords: Anti-HNE antibodies; DHN-MA, 1,4-Dihydroxynonane-mercapturic acid; DNPH, 2,4-Dinitrophenylhydrazine; ESI, Electrospray ionization; FT-ICR, Fourier transform ion cyclotron resonance; HNE, 4-Hydroxy-2-nonenal; HNE-protein adducts; HODA, 9-Hydroxy-12-oxo-10(E)-dodecenoic acid; HPETE, Hydroperoxyeicosatetraenoic acid; HPODE, Hydroperoxyoctadecadienoic acid; Hydroxyalkenal; KODA, 9-Keto-12-oxo-10(E)-dodecenoic acid; MALDI, Matrix assisted laser desorption ionization; MDA, Malondialdehyde; MS, Mass spectrometry; Mab, Monoclonal antibody; Mass spectrometry; Neutral loss scanning; ONA, 9-Oxo-nonanoic acid; ONE, 9-Oxo-2-nonenal; PETE, Peroxyeicosatetraenoate; PODE, Peroxyoctadecadienoate; Redox signalling.

Graphical abstract

Fig. 1

Generic mechanisms for cleavage of oxidized fatty acyl chains to yield HNE and ONA. (A) Protonation of the hydroperoxide yields a good leaving group and rearrangement of a C3C to C3O bond. The resulting carbonium ion is unstable, and hydrolysis occurs. (B) A peroxy radical cyclizes to form a dioxetane and carbon-centred radical. The dioxetane can rearrange by a 2-electron process resulting in cleavage of the dioxetane ring. The C-centred radical is susceptible to attack by oxygen to form a 2nd hydroperoxide, probably before fragmentation occurs. (C) In the presence of transition metals, hydroperoxides are converted to alkoxy radicals, which initiate a radical rearrangement resulting in β-scission.

Fig. 2

Formation of some intermediates on the pathways from linoleic acid (C18:2) to HNE. Analogous pathways exist for arachidonic acid (C20:4) with starting materials 11-hydroperoxy-eicosatetraenoic acid and 15-hydroperoxy-eicosatetraenoic acid.

Fig. 3

Overview of most common methods for detecting and analyzing HNE and its protein adducts.

Fig. 4

Schematic mass spectra showing how the formation of a Michael adduct at a histidine residue affects the fragmentation pattern of a peptide during MSMS sequencing by adding m/z 156 to the mass of the corresponding y₆ and y₇ fragment ions. The y ions are shown in brown; the grey peaks represent b ions or other minor fragment ions.