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.