Lipid Adducts Research Articles

Overcoming thermostability challenges in mRNA–lipid nanoparticle systems with piperidine-based ionizable lipids

Lipid nanoparticles (LNPs) have emerged as promising platforms for efficient in vivo mRNA delivery owing to advancements in ionizable lipids. However, maintaining the thermostability of mRNA/LNP systems remains challenging. While the importance of only a small amount of lipid impurities on mRNA inactivation is clear, a fundamental solution has not yet been proposed. In this study, we investigate an approach to limit the generation of aldehyde impurities that react with mRNA nucleosides through the chemical engineering of lipids. We demonstrated that piperidine-based lipids improve the long-term storage stability of mRNA/LNPs at refrigeration temperature as a liquid formulation. High-performance liquid chromatography analysis and additional lipid synthesis revealed that amine moieties of ionizable lipids play a vital role in limiting reactive aldehyde generation, mRNA–lipid adduct formation, and loss of mRNA function during mRNA/LNP storage. These findings highlight the importance of lipid design and help enhance the shelf-life of mRNA/LNP systems.

S-26-04 Structure-function studies of the effects of reactive oxidized lipid adducts on metabolic regulation

S-26-04 Structure-function studies of the effects of reactive oxidized lipid adducts on metabolic regulation

Biochemical Mechanisms Associating Alcohol Use Disorders with Cancers.

Simple SummaryOf all yearly deaths attributable to alcohol consumption globally, approximately 12% are due to cancers, representing approximately 0.4 million deceased individuals. Ethanol metabolism disturbs cell biochemistry by targeting the structure and function of essential biomolecules (proteins, nucleic acids, and lipids) and by provoking alterations in cell programming that lead to cancer development and cancer malignancy. A better understanding of the metabolic and cell signaling realm affected by ethanol is paramount to designing effective treatments and preventive actions tailored to specific neoplasias.The World Health Organization identifies alcohol as a cause of several neoplasias of the oropharynx cavity, esophagus, gastrointestinal tract, larynx, liver, or female breast. We review ethanol’s nonoxidative and oxidative metabolism and one-carbon metabolism that encompasses both redox and transfer reactions that influence crucial cell proliferation machinery. Ethanol favors the uncontrolled production and action of free radicals, which interfere with the maintenance of essential cellular functions. We focus on the generation of protein, DNA, and lipid adducts that interfere with the cellular processes related to growth and differentiation. Ethanol’s effects on stem cells, which are responsible for building and repairing tissues, are reviewed. Cancer stem cells (CSCs) of different origins suffer disturbances related to the expression of cell surface markers, enzymes, and transcription factors after ethanol exposure with the consequent dysregulation of mechanisms related to cancer metastasis or resistance to treatments. Our analysis aims to underline and discuss potential targets that show more sensitivity to ethanol’s action and identify specific metabolic routes and metabolic realms that may be corrected to recover metabolic homeostasis after pharmacological intervention. Specifically, research should pay attention to re-establishing metabolic fluxes by fine-tuning the functioning of specific pathways related to one-carbon metabolism and antioxidant processes.

Human Brain Lipidomics: Utilities of Chloride Adducts in Flow Injection Analysis.

Ceramides have been implicated in a number of disease processes. However, current means of evaluation with flow infusion analysis (FIA) have been limited primarily due to poor sensitivity within our high-resolution mass spectrometry lipidomics analytical platform. To circumvent this deficiency, we investigated the potential of chloride adducts as an alternative method to improve sensitivity with electrospray ionization. Chloride adducts of ceramides and ceramide subfamilies provided 2- to 50-fold increases in sensitivity both with analytical standards and biological samples. Chloride adducts of a number of other lipids with reactive hydroxy groups were also enhanced. For example, monogalactosyl diacylglycerols (MGDGs), extracted from frontal lobe cortical gray and subcortical white matter of cognitively intact subjects, were not detected as ammonium adducts but were readily detected as chloride adducts. Hydroxy lipids demonstrate a high level of specificity in that phosphoglycerols and phosphoinositols do not form chloride adducts. In the case of choline glycerophospholipids, the fatty acid substituents of these lipids could be monitored by MS2 of the chloride adducts. Monitoring the chloride adducts of a number of key lipids offers enhanced sensitivity and specificity with FIA. In the case of glycerophosphocholines, the chloride adducts also allow determination of fatty acid substituents. The chloride adducts of lipids possessing electrophilic hydrogens of hydroxyl groups provide significant increases in sensitivity. In the case of glycerophosphocholines, chloride attachment to the quaternary ammonium group generates a dominant anion, which provides the identities of the fatty acid substituents under MS2 conditions.

A Drastic Shift in Lipid Adducts in Colon Cancer Detected by MALDI-IMS Exposes Alterations in Specific K+ Channels.

Simple SummaryColorectal cancer (CRC) is one of the most preventable yet deadliest cancers, one reason being that it involves very different lesions. Currently, there is a great international effort to improve CRC classification using as many molecular features as possible. A cutting-edge technique, imaging mass spectrometry, is used to enable the visualization of the bidimensional (2D) distribution of molecules across tissues in order to study how the composition of the cell membrane, in particular membrane lipids, changes in tumors. Our previous studies indicate that lipid composition is highly sensitive to cell alterations. Importantly, during the analysis, we are also able to establish changes in charged lipids, observations that can be misinterpreted. A close study of our results alongside information from public databases leads to the identification of gene coding for a potassium channel that could account for our observations and could represent a suitable target for drug development.Even though colorectal cancer (CRC) is one of the most preventable cancers, it is one of the deadliest, and recent data show that the incidence in people <50 years has unexpectedly increased. While new techniques for CRC molecular classification are emerging, no molecular feature is as yet firmly associated with prognosis. Imaging mass spectrometry (IMS) lipidomic analyses have demonstrated the specificity of the lipid fingerprint in differentiating pathological from healthy tissues. During IMS lipidomic analysis, the formation of ionic adducts is common. Of particular interest is the [Na+]/[K+] adduct ratio, which already functions as a biomarker for homeostatic alterations. Herein, we show a drastic shift of the [Na+]/[K+] adduct ratio in adenomatous colon mucosa compared to healthy mucosa, suggesting a robust increase in K+ levels. Interrogating public databases, a strong association was found between poor diagnosis and voltage-gated potassium channel subunit beta-2 (KCNAB2) overexpression. We found this overexpression in three CRC molecular subtypes defined by the CRC Subtyping Consortium, making KCNAB2 an interesting pharmacological target. Consistently, its pharmacological inhibition resulted in a dramatic halt in commercial CRC cell proliferation. Identification of potential pharmacologic targets using lipid adduct information emphasizes the great potential of IMS lipidomic techniques in the clinical field.

Circulating Hedgehog: a fresh view of a classic morphogen.

Members of the Hedgehog family of morphogens mediate the intercellular communication necessary for the organisation and development of many animal tissues. They are modified by various lipid adducts, rendering them insoluble in hydrophilic environments and leading to the contentious question of how these molecules travel in the aqueous extracellular space. Seminal work carried out by Suzanne Eaton and her colleagues has shed light on how these morphogens can spread over long distances through their association with lipoprotein particles. In this Spotlight article, we discuss Suzanne's pioneering work and her contribution to our understanding of the transport and activity of morphogens, in particular Hedgehog. We also describe two other essential aspects of her work: the discovery and characterisation of endogenously present Hedgehog variants, as well as her proposition that, in addition to its role as a morphogen, Hedgehog acts as an endocrine hormone.

Functions of Wnt and Hedgehog-containing extracellular vesicles in development and disease.

Secreted morphogens play a major role in the intercellular communication necessary for animal development. It was initially thought that, in order to organize tissue morphogenesis and control cell fate and proliferation, morphogens diffused freely in the extracellular space. This view has since changed following the discovery that morphogens of the Wnt and Hedgehog (Hh) families are modified by various lipid adducts during their biosynthesis, providing them with high affinity for the membrane bilayer. Recent work performed in model organisms suggests that Wnt and Hh proteins are carried on extracellular vesicles. In this Review, we provide our perspectives on the mechanisms of formation of Wnt- and Hh-containing extracellular vesicles, and discuss their functions during animal development, as well as in various human physiopathologies.

Changes in Protein Fluorescence in a Lipid-Protein Co-oxidizing Oleogel.

High and low levels of lipid-induced protein oxidation (tuned by the addition of 0%-8.4% water) were investigated in oleogels, using excitation-emission matrix (EEM) fluorescence spectroscopy, coupled with a partial least-squares (PLS) regression and lipid hydroperoxide data. In high-level oxidation models, the intrinsic tryptophan fluorescence decreased and the emission maxima increased from 352.5 to 356.0 nm indicating the presence of protein modifications, which was further supported by size-exclusion chromatography. PLS recognized 3 latent components, with several excitation-emission points of interest. These apparent compounds include a region associated with radical mediated protein modifications (approximately 325 and 410 nm), lipid oxidation product adducts (approximately 350 nm and 420-425 nm), and malondialdehyde adducts (approximately 375 and 425 nm). The separate evaluation of these apparent compounds, at a 420 nm emission, indicated that lipid oxidation promotes protein lipid adduct fluorescence at high water levels, rather than radical mediated protein fluorescence.

High-Performance Molecular Imaging with MALDI Trapped Ion-Mobility Time-of-Flight (timsTOF) Mass Spectrometry.

Imaging mass spectrometry (IMS) enables the spatially targeted molecular assessment of biological tissues at cellular resolutions. New developments and technologies are essential for uncovering the molecular drivers of native physiological function and disease. Instrumentation must maximize spatial resolution, throughput, sensitivity, and specificity, because tissue imaging experiments consist of thousands to millions of pixels. Here, we report the development and application of a matrix-assisted laser desorption/ionization (MALDI) trapped ion-mobility spectrometry (TIMS) imaging platform. This prototype MALDI timsTOF instrument is capable of 10 μm spatial resolutions and 20 pixels/s throughput molecular imaging. The MALDI source utilizes a Bruker SmartBeam 3-D laser system that can generate a square burn pattern of <10 × 10 μm at the sample surface. General image performance was assessed using murine kidney and brain tissues and demonstrate that high-spatial-resolution imaging data can be generated rapidly with mass measurement errors <5 ppm and ∼40 000 resolving power. Initial TIMS-based imaging experiments were performed on whole-body mouse pup tissue demonstrating the separation of closely isobaric [PC(32:0) + Na]+ and [PC(34:3) + H]+ (3 mDa mass difference) in the gas phase. We have shown that the MALDI timsTOF platform can maintain reasonable data acquisition rates (>2 pixels/s) while providing the specificity necessary to differentiate components in complex mixtures of lipid adducts. The combination of high-spatial-resolution and throughput imaging capabilities with high-performance TIMS separations provides a uniquely tunable platform to address many challenges associated with advanced molecular imaging applications.

CD39 and CD73 activity are protective in a mouse model of antiphospholipid antibody-induced miscarriages

ObjectiveAntiphospholipid syndrome (APS) is a systemic autoimmune disorder of young adults associated with devastating pregnancy complications (recurrent miscarriages, preeclampsia and low birth weight) and vascular complications including thrombosis. The key components implicated in pathogenesis of APS are the complement cascade and tissue factor (TF) activity causing inflammation and coagulation. Purinergic signalling involving catabolism of ATP to adenosine by cell-surface enzymes CD39 and CD73 has anti-inflammatory and anti-thrombotic effects. We studied whether activities of CD39 and CD73 are important in preventing the development of miscarriages in APS. MethodsWe studied frequency of miscarriages and decidual pathology following passive transfer of human aPL-ab to pregnant wildtype mice, and mice deficient in CD39 and CD73, and also transgenic mice exhibiting 2-3X higher CD39 activity. ResultsaPL-ab infusion in pregnant CD39-or CD73-knockout mice triggers an increase in miscarriages, associated with increased TF expression and complement deposition as well as elevated oxidative stress and pro-inflammatory TNF-α and IL-10 expression within the placental decidua. In contrast, aPL-ab induced miscarriages are prevented in mice over-expressing CD39, with reduced decidual TF expression and C3d deposition, diminished lipid peroxidation (4-hydroxynonenal or 4-HNE positive lipid adducts), and reduced TNF-α expression. ConclusionWe demonstrate a protective role for CD39 in APS and provide rationale for both the development of endothelial cell-targeted soluble CD39 as a novel therapeutic for APS and analysis of perturbations in the purinergic pathway to explain human disease.

Ozone-induced dissociation on a traveling wave high-resolution mass spectrometer for determination of double-bond position in lipids.

The position of C=C within fatty acyl chains affects the biological function of lipids. Ozone-induced dissociation mass spectrometry (OzID-MS) has great potential in determination of lipid double-bond position, but has generally been implemented on low-resolution ion trap mass spectrometers. In addition, most of the OzID-MS experiments carried out so far were focused on the sodiated adducts of lipids; fragmentation of the most commonly observed protonated ions generated in LC/MS-based lipidomics workflow has been less explored. Ozone generated in line from an ozone generator was connected to the trap and transfer gas supply line of a Synapt G2 high-resolution mass spectrometer. Protonated ions of different phosphatidylcholines (PC) were generated by electrospray ionization through direct infusion. Different parameters, including traveling wave height and velocity, trap entrance and DC potential, were adjusted to maximize the OzID efficiency. sn-positional isomers and cis/trans isomers of lipids were compared for their reactivity with ozone. Traveling wave height and velocity were tuned to prolong the encounter time between lipid ions and ozone, and resulted in improved OzID efficiency, as did increasing trapping region DC and entrance potential. Under optimized settings, at least 1000 times enhancement in OzID efficiency was achieved compared to that under default settings for monounsaturated PC standards. Monounsaturated C=C in the sn-2 PC isomer reacted faster with ozone than the sn-1 isomer. Similarly, the C=C in trans PC reacted faster than in cis PC. This is the first implementation of OzID in the trap and transfer region of a traveling wave enabled high-resolution mass spectrometer. The OzID reaction efficiency is significantly improved by slowing down ions in the trap region for their prolonged interaction with ozone. This will facilitate application of high-resolution OzID-MS in lipidomics.

Effects of Detergent Micelles on Lipid Binding to Proteins in Electrospray Ionization Mass Spectrometry.

A wide variety of biological processes rely upon interactions between proteins and lipids, ranging from molecular transport to the organization of the cell membrane. It was recently established that electrospray ionization mass spectrometry (ESI-MS) is capable of capturing transient interactions between membrane proteins and their lipid environment, and a detailed understanding of the underlying processes is therefore of high importance. Here, we apply ESI-MS to investigate the factors that govern complex formation in solution and gas phases by comparing nonselective lipid binding with soluble and membrane proteins. We find that exogenously added lipids did not bind to soluble proteins, suggesting that lipids have a low propensity to form electrospray ionization adducts. The presence of detergents at increasing micelle concentrations, on the other hand, resulted in moderate lipid binding to soluble proteins. A direct ESI-MS comparison of lipid binding to the soluble protein serum albumin and to the integral membrane protein NapA shows that soluble proteins acquire fewer lipid adducts. Our results suggest that protein–lipid complexes form via contacts between proteins and mixed lipid/detergent micelles. For soluble proteins, these complexes arise from nonspecific contacts between the protein and detergent/lipid micelles in the electrospray droplet. For membrane proteins, lipids are incorporated into the surrounding micelle in solution, and complex formation occurs independently of the ESI process. We conclude that the lipids in the resulting complexes interact predominantly with sites located in the transmembrane segments, resulting in nativelike complexes that can be interrogated by MS.

Tobacco nitrosamines as culprits in disease: mechanisms reviewed.

The link between tobacco abuse and cancer is well-established. However, emerging data indicate that toxins in tobacco smoke cause cellular injury due to enhanced toxic/metabolic effects of metabolites, disruption of intracellular signaling mechanisms, and formation of DNA, protein, and lipid adducts that impair function and promote oxidative stress and inflammation. These effects of smoking, which are largely non-carcinogenic, can be produced by tobacco-specific nitrosamines and their metabolites. These factors could account for the increased rates of neurodegeneration and insulin resistance diseases among smokers. Herein, we review nicotine and tobacco-specific nitrosamine metabolism, mechanisms of adduct formation, DNA damage, mutagenesis, and potential mechanisms of disease.

Methanothermobacter thermautotrophicus modulates its membrane lipids in response to hydrogen and nutrient availability.

Methanothermobacter thermautotrophicus strain ΔH is a model hydrogenotrophic methanogen, for which extensive biochemical information, including the complete genome sequence, is available. Nevertheless, at the cell membrane lipid level, little is known about the responses of this archaeon to environmental stimuli. In this study, the lipid composition of M. thermautotrophicus was characterized to verify how this archaeon modulates its cell membrane components during growth phases and in response to hydrogen depletion and nutrient limitation (potassium and phosphate). As opposed to the higher abundance of phospholipids in the stationary phase of control experiments, cell membranes under nutrient, and energy stress were dominated by glycolipids that likely provided a more effective barrier against ion leakage. We also identified particular lipid regulatory mechanisms in M. thermautotrophicus, which included the accumulation of polyprenols under hydrogen-limited conditions and an increased content of sodiated adducts of lipids in nutrient-limited cells. These findings suggest that M. thermautotrophicus intensely modulates its cell membrane lipid composition to cope with energy and nutrient availability in dynamic environments.

P93 - Targeted mass spectrometry methods for detecting oxidative post-translational modifications

Oxidative post-translational modifications (oxPTMs) can alter the function of proteins, and are important in the redox regulation of cell behaviour. The most informative technique to detect and locate oxPTMs within proteins is mass spectrometry (MS). However, proteomic MS data are usually searched against theoretical databases using statistical search engines, and the occurrence of unspecified or multiple modifications, or other unexpected features, can lead to failure to detect the modifications and erroneous identifications of oxPTMs. We have developed a new approach for mining data from accurate mass instruments that allows multiple modifications to be examined. Accurate mass extracted ion chromatograms (XIC) for specific reporter ions from peptides containing oxPTMs were generated from standard LC-MSMS data acquired on a rapid-scanning high-resolution mass spectrometer (ABSciex 5600 Triple TOF). The method was tested using proteins from human plasma or isolated LDL. A variety of modifications including chlorotyrosine, nitrotyrosine, kynurenine, oxidation of lysine, and oxidized phospholipid adducts were detected. For example, the use of a reporter ion at 184.074Da/e, corresponding to phosphocholine, was used to identify for the first time intact oxidized phosphatidylcholine adducts on LDL. In all cases the modifications were confirmed by manual sequencing. ApoB-100 containing oxidized lipid adducts was detected even in healthy human samples, as well as LDL from patients with chronic kidney disease. The accurate mass XIC method gave a lower false positive rate than normal database searching using statistical search engines, and identified more oxidatively modified peptides. A major advantage was that additional modifications could be searched after data collection, and multiple modifications on a single peptide identified. The oxPTMs present on albumin and ApoB-100 have potential as indicators of oxidative damage in ageing or inflammatory diseases.

Toll-Like Receptor 2-Dependent Protection against Pneumococcal Carriage by Immunization with Lipidated Pneumococcal Proteins

Infections with Streptococcus pneumoniae cause substantial morbidity and mortality, particularly in children in developing nations. Polysaccharide-conjugate vaccines provide protection against both invasive disease and colonization, but their use in developing countries is limited by restricted serotype coverage and expense of manufacture. Using proteomic screens, we recently identified several antigens that protected mice from pneumococcal colonization in a CD4(+) T cell- and interleukin-17A (IL-17A)-dependent manner. Since several of these proteins are lipidated, we hypothesized that their immunogenicity and impact on colonization are in part due to activation of Toll-like receptor 2 (TLR2), a receptor for lipoproteins. Here we show that lipidated versions of the antigens elicited significantly higher activation of both human embryonic kidney cells engineered to express TLR2 (HEK-TLR2) and wild-type (WT) murine macrophages than nonlipidated mutant antigens. Lipoprotein-stimulated secretion of proinflammatory cytokines was ∼10× to ∼100× lower in murine TLR2-deficient macrophages than in WT macrophages. Subcutaneous immunization of C57BL/6 mice with protein subunit vaccines containing one or two of these lipoproteins or protein fusion constructs bearing N-terminal lipid adducts elicited a robust IL-17A response and a significant reduction in colonization compared with immunization with alum alone. In contrast, immunization of Tlr2(-/-) mice elicited no detectable IL-17A response and no protection against pneumococcal colonization. These experiments suggest that the lipid moieties enhance the immunogenicity and protective efficacy of pneumococcal TH17 antigens through activation of TLR2. Thus, triggering TLR2 with an antigen-specific protein subunit formulation is a possible strategy for the development of a serotype-independent pneumococcal vaccine that would reduce pneumococcal carriage.

Dynamic Association with Donor Cell Filopodia and Lipid-Modification Are Essential Features of Wnt8a during Patterning of the Zebrafish Neuroectoderm

BackgroundWnt proteins are conserved signaling molecules that regulate pattern formation during animal development. Many Wnt proteins are post-translationally modified by addition of lipid adducts. Wnt8a provides a crucial signal for patterning the anteroposterior axis of the developing neural plate in vertebrates. However, it is not clear how this protein propagates from its source, the blastoderm margin, to the target cells in the prospective neural plate, and how lipid-modifications might influence Wnt8a propagation and activity.ResultsWe have dynamically imaged biologically active, fluorescently tagged Wnt8a in living zebrafish embryos. We find that Wnt8a localizes to membrane-associated, punctate structures in live tissue. In Wnt8a expressing cells, these puncta are found on filopodial cellular processes, from where the protein can be released. In addition, Wnt8a is found colocalized with Frizzled receptor-containing clusters on signal receiving cells. Combining in vitro and in vivo assays, we compare the roles of conserved Wnt8a residues in cell and non-cell-autonomous signaling activity and secretion. Non-signaling Wnt8 variants show these residues can regulate Wnt8a distribution in producing cell membranes and filopodia as well as in the receiving tissue.ConclusionsTogether, our results show that Wnt8a forms dynamic clusters found on filopodial donor cell and on signal receiving cell membranes. Moreover, they demonstrate a differential requirement of conserved residues in Wnt8a protein for distribution in producing cells and receiving tissue and signaling activity during neuroectoderm patterning.

Hedgehog Pathway Modulation by Multiple Lipid Binding Sites on the Smoothened Effector of Signal Response

Hedgehog (Hh) signaling during development and in postembryonic tissues requires activation of the 7TM oncoprotein Smoothened (Smo) by mechanisms that may involve endogenous lipidic modulators. Exogenous Smo ligands previously identified include the plant sterol cyclopamine (and its therapeutically useful synthetic mimics) and hydroxylated cholesterol derivatives (oxysterols); Smo is also highly sensitive to cellular sterol levels. The relationships between these effects are unclear because the relevant Smo structural determinants are unknown. We identify the conserved extracellular cysteine-rich domain (CRD) as the site of action for oxysterols on Smo, involving residues structurally analogous to those contacting the Wnt lipid adduct in the homologous Frizzled CRD; this modulatory effect is distinct from that of cyclopamine mimics, from Hh-mediated regulation, and from the permissive action of cellular sterol pools. These results imply that Hh pathway activity is sensitive to lipid binding at several Smo sites, suggesting mechanisms for tuning by multiple physiological inputs.

Glucosepane: a poorly understood advanced glycation end product of growing importance for diabetes and its complications

Advanced glycation end products (AGEs) represent a family of protein, peptide, amino acid, nucleic acid and lipid adducts formed by the reaction of carbonyl compounds derived directly or indirectly from glucose, ascorbic acid and other metabolites such as methylglyoxal. AGE formation in diabetes is of growing importance for their role as markers and potential culprits of diabetic complications, in particular retinopathy, nephropathy and neuropathy. Development of sensitive and specific assays utilizing liquid chromatography mass spectrometry with isotope dilution method has made it possible to detect and quantitate non-UV active AGEs such as carboxymethyl-lysine and glucosepane, the most prevalent AGE and protein crosslink of the extracellular matrix. Below we review studies on AGE formation in two skin biopsies obtained near the closeout of the Diabetes Control and Complications Trial (DCCT), one of which was processed in 2011 for assay of novel AGEs. The results of these analyses show that while several AGEs are associated and predict complication progression, the glucose/fructose-lysine/glucosepane AGE axis is one of the most robust markers for microvascular disease, especially retinopathy, in spite of adjustment for past or future average glycemia. Yet overall little biological and clinical information is available on glucosepane, making this review a call for data in a field of growing importance for diabetes and chronic metabolic diseases of aging.

Pro‐ and Antioxidative Effect of α‐Tocopherol on Edible Oils, Triglycerides and Fatty Acids

Using advanced electron paramagnetic resonance techniques (EPR), oxidation of crude vegetable oils and their components (fatty acids and triglycerides) by radicals generated from hydrogen peroxide was investigated. The correlation rotational times were determined allowing us to characterize radicals formed during edible oils oxidation. Additionally 1H- and 14N-hyperfine coupling constants differentiate the fatty acids dependently on their unsaturation. The acids with a higher number of unsaturated bonds exhibit higher AN values of PBN/·lipid adduct. The waste oil with high free fatty acids content underwent the oxidation reaction more efficiently, however due to saturation and the high content of the fatty acids the carbon-centered radicals formed (upon hydrogen peroxide radicals) and their PBN (N-tert-butyl-α-phenylnitrone) adducts were less stable. The antioxidant effect was dependent on the amount of α-tocopherol added. In small amounts of up to 0.35 mg/1 g of fatty acid or triglyceride, it inhibited the creation of PBN/·lipid adducts while with higher amounts it intensified adduct formation. The α-tocopherol (AT) addition influence was also studied as spin scavenging dependence and indicated that any addition of the antioxidant in the investigated samples led to free radical scavenging and the effect increased with the increase in AT content.