Regular ArticleEffects of oxidation on the physicochemical properties of polyunsaturated lipid membranes
Graphical abstract
Introduction
Many cellular processes and pathological conditions such as apoptosis, inflammation, infection, or sepsis, are modulated or triggered by oxidative stress through direct damage of the cell membrane [1]. Both enzymatic and non-enzymatic oxidation, the latter occurring via direct exposure to reactive oxygen species (ROS), lead to lipid peroxidation in living cells and tissues. The exposure of biological membranes to ROS results in a complex mixture of oxidized phospholipids (OxPL), including peroxyl radicals, hydroperoxides, truncated phospholipids, and acyl chain fragments such as carbonyl compounds [2]. These OxPL are not just by-products of lipid peroxidation but also active modulators of signaling processes, e.g., initiating and amplifying inflammation, and also acting as danger markers for damaged tissue [3], [4]. For instance, OxPL, and particularly oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), play an active role in the onset of atherosclerosis [5]. PAPC is one of the most abundant polyunsaturated phospholipids in mammalian tissues [6], and even low doses of its oxidized form strongly affect the permeability of endothelial cell layers, enhancing low-density lipoprotein (LDL) entry and deposition into blood vessels. PAPC also activates several pathways related to oxidation and inflammation, such as the synthesis and release of growth factors, cytokines, and chemokines [5], [7]. Many previous studies of membrane oxidation have focused on monounsaturated or saturated lipids; however, polyunsaturated fatty acids (PUFA) are much more susceptible to oxidation as they contain several methylene groups located between double bonds, known as bisallylic groups, characterized by weaker CH bonds and so more prompt to H extraction [2]. Therefore, the inclusion of polyunsaturated phospholipids into membrane oxidation models is crucial for modelling, characterizing, and understanding the complex processes occurring in real mammalian membranes and tissues.
As shown in earlier studies, photogeneration of oxidative stress, e.g., by shortwave (λ ∼ 250 nm) ultraviolet (UV) irradiation, is a convenient way of controlling oxidation levels in different model lipid membrane systems [8]. The most common free radicals produced in aqueous media under oxidative conditions are singlet oxygen () and hydroxyl radicals (OH). After exposure of PUFA-containing membranes to ROS, in the presence of oxygen, peroxyl radicals are produced, which are subsequently transformed into hydroperoxides by reaction with other PUFA molecules. Following initiation, oxidation continues via an enzyme-independent process, resulting in a wide spectrum of OxPLs. This chain reaction, known as lipid peroxidation, continues until two radicals combine to form a stable product, or until the radicals are neutralized by a chain breaking antioxidant [2]. Final products include truncated phospholipids, which have lost some or all bisallylic methylene groups, and lipids with cyclized acyl chains. Inclusion of these lipid oxidation products has been found to change the lateral structure [9], bilayer stability and membrane permeability [10], [11], as well as to affect lipid-protein interactions [12].
Scattering techniques, particularly small-angle X-ray scattering (SAXS) and neutron reflectometry (NR), are useful tools for the characterization of lipid membrane oxidation due to their high sensitivity to the phase behaviour and structural rearrangements of lipid membranes [13], [14]. For instance, SAXS has been used to study micro- and nanoscopic 2D phase separated bilayers [15]. The effects of lipid peroxidation on the structure and organization of model PC liposomes have been previously studied by small-angle X-ray diffraction, and reductions in the bilayer thickness and potential interdigitation under oxidative stress reported [16], along with enhanced lateral phase separation in the presence of cholesterol [17]. NR has been used to study changes of supported lipid bilayers through lipid exchange, removal or degradation by exploitation of contrast variation, i.e., by adjusting the H2O/D2O ratio of the surrounding medium to highlight different parts of the bilayer [18], [19], [20]. However, despite the capacity of NR to detect compositional and structural changes at the Ångstrom level, such as phospholipid degradation or structural rearrangements, to date only a few studies have addressed oxidative degradation of lipid membranes. For instance, the oxidation of ozone-exposed lipid monolayers [21], [22] or UV-exposed supported bilayers [8], [10], [20] has been investigated using NR. These studies have focused on non- or slightly-oxidizable compositions, using lipids with saturated or monounsaturated acyl chains, such as palmitoyl or oleyl chains. In contrast, the oxidation of lipids containing PUFAs, significantly more prone to oxidation and very abundant in biological tissues, have not yet been explored using neutrons.
In this work, we therefore aim to investigate the effects of oxidative stress on the structure and stability of membranes of varying content of polyunsaturated lipids, using simple mixtures of palmitoyloleoylphosphatidylcholine (POPC) and PAPC. Lipid peroxidation was triggered by shortwave UV exposure, with or without the addition of peroxidation initiator and propagator, H2O2. These models were then investigated using a battery of biophysical and surface-chemistry techniques, including NR, SAXS, Fourier transform infrared spectroscopy with attenuated total reflection (FTIR-ATR), fluorescence spectroscopy, and light scattering, in order to obtain an overall view on structural, compositional, and stability changes of polyunsaturated phospholipid bilayers under oxidative stress.
Section snippets
Materials
The phospholipids investigated here, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (16:0–18:1 PC, POPC) and 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (16:0–22:4 PC, PAPC), both of >99% purity, were obtained from Avanti Polar Lipids (Alabaster, USA). C11-BODIPY 581/591 was purchased from Molecular Probes/Thermo Fisher Scientific (USA). Calcein (Bis[N,N-bis(carboxymethyl)aminomethyl]fluorescein), Tris buffer (Trizma® base, ≥99.9%), D2O (>99.9%), sodium chloride (>99.5%), and all
Comparison between oxidative treatments in terms of lipid peroxidation
As a first step, a comparison between different physical treatments proposed in literature for oxidizing lipids, including shortwave UV irradiation, autooxidation by heat (60 °C), and tip probe sonication, was carried out using POPC:PAPC LUVs. These tests were performed using a standard thiobarbituric acid reactive substances (TBARS) assay, in which lipid oxidation products such as malondialdehyde (MDA) react with thiobarbituric acid to form a coloured and fluorescent product. From these
Discussion
In the present study, we have described how the degree of polyunsaturation influences bilayer oxidation, with consequences on thickness, lateral structure, and permeability. As observed by NR, there is a reduction in the stability of POPC:PAPC bilayers with increasing PAPC content. In addition, as confirmed by neutron and X-ray scattering, bilayer thicknesses are reduced, connected to corresponding increases in APM. These results are consistent with a body of literature that has characterized
Conclusions
The present investigation addresses effects of polyunsaturation on lipid oxidation, and consequences of this for membrane structure and stability. The combined use of X-ray scattering and neutron reflectometry, FTIR-ATR, DLS, and fluorescence spectroscopy demonstrates a richness in membrane oxidation effects, regardless of the simplicity of the POPC:PAPC system investigated here. Oxidation of these polyunsaturated lipid membranes results in a combination of structural, compositional, and
Acknowledgements
Financial support is acknowledged from the LEO Foundation Center for Cutaneous Drug Delivery (grant number 2016-11-01; KLM, LSED, and MM), the Novo Nordisk Foundation Interdisciplinary Synergy program SYNERGY (grant number NNF15OC0016670; SB), and travel funding for NR experiments from the Danish Natural Sciences Research Council (DanScatt). Furthermore, beamtime at ILL (DOI: 10.5291/ILL-DATA.9-13-733) is gratefully acknowledged. In addition, we would like to thank Dr. Giovanna Fragneto for
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