1 Introduction

Lipid is an important component of food and the carrier of fat-soluble substances, which can not only provide heat and essential fatty acids, but can also enhance the flavor of food (Coca et al., 2011). Lipid oxidation is very common in the food system, especially during food processing. Various aldehydes are generated as secondary products of lipid oxidation. Compared with free radicals, these aldehydes are relatively stable and have a longer lifetime. Therefore, they can diffuse from the site of formation and migrate over long distances to react with various biological macromolecules (e.g, protein), thereby acting as ”toxic second messengers” of lipid oxidation (Hidalgo et al., 2017; Wu et al., 2009).
The main hazard of these active aldehydes is that they can react with nucleophilic macromolecules such as proteins, resulting in protein aggregation, dysfunction, immunogenicity, and activation of specific receptors (Colzani et al., 2016). Protein modification by aldehydes is generally considered to be closely related to formation of lipofuscin, which is associated with aging, and play a significant role in the onset and progression of various chronic diseases, such as neurodegenerative and cardiovascular diseases(Colzani et al., 2013).
Among the aldehydes with high reactivity, the most extensively studied were acrolein (ACR), 4-hydroxy-2-alkenals, and malondialdehyde (MDA). Today, 4,5-epoxy-2-alkenals and 4-oxo-2-alkenal have become a hot research topic (Hidalgo et al., 2000).
Among the active aldehydes generated from unsaturated fatty acids, there is also a large number of other aldehydes in addition to those mentioned above. The oil in unsaturated fatty acid produced a variety of unsaturated aldehydes under hot working conditions. One such example is E,E-2,4-alkadienals with 6-10 carbon atoms (Guillén et al.,2012; Hidalgo et al.,2016). Oxidation of n-6 series of polyunsaturated fatty acids (such as linoleic acid) was found to produce a large number of E,E-2,4-decadienal (Poyato et al., 2014; Sousa et al., 2017); however, oxidation of linolenic acid and its esters produced E,E-2,4-heptadienal, especially under heat stress (Hidalgo et al., 2016; Poyato et al., 2014). Some unsaturated aldehydes with a large number of carbon atoms without oxygen-containing side chains have relatively high molecular weight, which results in lower reaction activity than the widely studied acrolein (ACR), which has a small molecular weight and is a 4-hydroxy-trans-2-nonenal (HNE) with a hydroxyl side chain. Therefore, these unsaturated aldehydes have received relatively low attention (Sousa et al., 2017). Protein modification by these aldehydes have rarely been studied, and chemical reactions between these aldehydes and protein nucleophiles are not completely clear.
Previous reports have shown that long-chain aldehydes with oxygen-containing side chains (e.g, HNE, ONE et al) and short chain side aldehydes with no oxygen side chain (e.g ACR, Butenal et al) can cause significant protein damage. This prompted us to investigate whether modification by long-chain unsaturated aldehydes without oxygen-containing side chains have any effects on proteins, including how their chain length and concentration affect the degree of protein modification, and what the characteristics of these aldehydes are after binding with proteins.
While in vivo oxidative damage is a long term and cumulative process, most of the in vitro research are short-term studies. Therefore, the concentration of in vitro modifiers are usually much higher as compared with that in vivo . Although there are some differences with the real system, this procedure is common in scientific literature when an in vitro protein modification is analyzed (Traverso et al.,2004) . In addition, the focus of basic research is to discover all possible changes (and mechanisms), which will be different from those that occur in nature. We have reviewed a large number of studies that examined the interaction between protein and aldehydes produced by lipid oxidation. We found that the concentration range of aldehydes added in these experiments is 0.01-100 mm. Taking into account all past in vitro experiments in literature, as well as own pre-experiment pilot studies, the concentration range of the three aldehydes we used was set to be 1-50 mM.
The objective of this study was to characterize the changes produced in BSA incubated in different concentrations of heptadienal, nonadienal and decadienal. A variety of methods, such as fluorescence, UV visible absorption, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and colorimetric analyses were combined to characterize the aldehyde-protein adducts obtained. The results of this work extended findings from other studies, demonstrating that aldehydes from lipid oxidation can contribute significantly to protein damage. Our findings should be help define specific roles of long-chain unsaturated aldehydes without oxygen-containing side chains from lipid oxidation in the formation of degenerative proteins. This can provide a basis for a deeper understanding of the effect of these modified proteins on cells.