Background

When polyunsaturated fatty acids (PUFAs) become oxidized in cell membranes, there are only two possible outcomes: 1) resulting lipid peroxides (LOOHs) are neutralized via enzyme-dependent (e.g., glutathione peroxidase-4, GPx4) and/or spontaneous reaction with redox-active molecules (e.g., quinones); or 2) the LOOHs degrade into highly reactive aldehydes, capable of forming adducts with proteins, DNA and other lipids. One of the more common aldehydes formed is 4-hydroxynonenal (HNE), an α, β-unsaturated aldehyde formed from peroxidation of n-6 PUFAs such as arachidonic acid and linoleic acid, both of which are abundant in phospholipid cell membranes. Accumulation of HNE and other related biogenic aldehydes, such as malondialdehyde, react with nucleophilic side chains in proteins and polypeptides to form stable protein adducts that can sometimes act as ‘toxic second messengers’ of oxidative stress. These reactions constitute a form of oxidative stress referred to as carbonyl stress. Hydroxynonenal can noncompetitively inhibit the activity of antioxidant enzymes like aldehyde dehydrogenase (ALDH2) and aldo-ketoreductase (AKR), further exacerbating accumulation and cytotoxicity of reactive carbonyl species (Jinsmaa, et al., 2009). Since LOOHs are notoriously difficult to directly measure in vivo, HNE-adducts serve as a surrogate biomarker of LOOHs, and the corresponding carbonyl stress imposed by them in cells and tissues have been reported in both experimental models and clinical studies of degenerative and age-related pathologies including Parkinson’s Disease, obesity and diabetes, cardiovascular diseases, and many cancers (Markesbery and Lovell, 1998; Orioli et al.; 1998; Selley, 1998; Traverso et al., 1998; Frohnert et al., 2011; Anderson et al., 2014; Katunga et al., 2015a; Anderson et al., 2018). Hydroxynonenal tends to react with nucleophilic moieties, such as the side-chains of cysteine, lysine, and histidine, either the carbonyl group, forming a Schiff base, or the β-carbon, forming a Michael addition adduct.

A variety of assays for HNE-adduct detection are commercially available. Most of them employ an ELISA method, using a proprietary combination of antigen-capture or ‘sandwich-ELISA’ formulation. These assays can vary widely in sensitivity, leading to problems with reproducibility and data compatibility across laboratories. Here we present a facile, economically conservative and reproducible assay for determining HNE-adduct levels in serum, cell-, and tissue-lysates.