Reactive Lipid Oxidation Products Research Articles

Novel Urinary Metabolites of Aldehydic Lipid Oxidation Products From Heated Soybean Oil Revealed by Aldehyde Abatement and Metabolomic Fingerprinting

ObjectivesHeated cooking oils are a constitutive component of contemporary human diets and a common ingredient in animal feed. Aldehydes, as the most reactive lipid oxidation products (LOPs) in heated oils, are widely considered as a contributor to the adverse health effect associated with heated oil consumption. Aldehydes-derived metabolites in biofluids are valuable for monitoring the exposure of the aldehydes from heated oils, but the efforts for their identification were hampered by the high reactivity of aldehydes as well as the chemical complexity caused by the coexistence of other LOPs in heated oils. To address this challenge, this study investigated the aldehydes-derived metabolites through the metabolomic fingerprinting of mouse urine samples from feeding heated oils with and without aldehyde abatement. MethodsThe heated soybean oil (HSO) was prepared by heating the control soybean oil (CSO) at 185°C for 6 h with constant air flow (50 ml/min). Both HSO and CSO were then mixed with the silica gel with piperazine side chain to remove their aldehyde content, producing the silica gel-treated HSO (HSO-si) and the silica gel-treated CSO (CSO-si), respectively. The urine, serum, fecal samples were collected from the C57BL/6 mice fed with 4 diets containing CSO, CSO-si, HSO, HSO-si, and then analyzed by the liquid chromatography-mass spectrometry (LC-MS) metabolomic analysis. ResultsThe silica gel treatment dramatically reduced the aldehyde contents in HSO. Novel urinary metabolites formed by the reactions between 2,4-decadienal and lysine were identified through the metabolomic comparison between the HSO samples and the samples from 3 other treatment groups. ConclusionsThe identification of novel urinary metabolites of aldehydic LOPs warrants further biochemical examination on the chemical and metabolic processes responsible for their formation. These metabolites could become the biomarkers for monitoring the exposure of aldehydes in humans and animals. Funding SourcesThe research is partially supported by the NIFA project MIN-18–125.

Abstract MP01: Deacetylation Of Mitochondrial Cyclophilin D K166 Inhibits Cytokine-induced Oxidative Stress And Attenuates Hypertension

We have previously reported that depletion Cyclophilin D (CypD), a regulatory subunit of mitochondrial permeability transition pore, improves vascular function and attenuates hypertension, however, specific regulation of CypD in hypertension is not clear. Analysis of human arterioles from hypertensive patients did not reveal alterations in CypD levels but showed 3-fold increase in CypD acetylation. We hypothesized that CypD-K166 acetylation promotes vascular oxidative stress and hypertension, and measures to reduce CypD acetylation can improve vascular function and reduce hypertension. Essential hypertension and animal models of hypertension are linked to inactivation of mitochondrial deacetylase Sirt3 by highly reactive lipid oxidation products, isolevuglandins (isoLGs), and supplementation of mice with mitochondria targeted scavenger of isoLGs, mito2HOBA, improves CypD deacetylation. To test the specific role of CypD-K166 acetylation, we developed CypD-K166R deacetylation mimic mutant mice. Mitochondrial respiration, vascular function and systolic blood pressure in CypD-K166R mice was similar to wild-type C57Bl/6J mice. Meanwhile, angiotensin II-induced hypertension was substantially attenuated in CypD-K166R mice (144 mmHg) compared with wild-type mice (161 mmHg). Angiotensin II infusion in wild-type mice significantly increased mitochondrial superoxide, impaired endothelial dependent relaxation, and reduced the level of endothelial nitric oxide which was prevented in angiotensin II-infused CypD-K166R mice. Hypertension is linked to increased levels of inflammatory cytokines TNFα and IL-17A promoting vascular oxidative stress and end-organ damage. We have tested if CypD-K166R mice are protected from cytokine-induced oxidative stress. Indeed, ex vivo incubation of aorta with the mixture of angiotensin II, TNFα and IL-17A (24 hours) increased mitochondrial superoxide by 2-fold in wild-type aortas which was abrogated in CypD-K166R mice. These data support the pathophysiological role of CypD acetylation in inflammation, oxidative stress and hypertensive end-organ damage. We propose that targeting CypD acetylation may have therapeutic potential in treatment of vascular dysfunction and hypertension.

Modification of proteins by reactive lipid oxidation products and biochemical effects of lipoxidation.

Lipid oxidation results in the formation of many reactive products, such as small aldehydes, substituted alkenals, and cyclopentenone prostaglandins, which are all able to form covalent adducts with nucleophilic residues of proteins. This process is called lipoxidation, and the resulting adducts are called advanced lipoxidation end products (ALEs), by analogy with the formation of advanced glycoxidation end products from oxidized sugars. Modification of proteins by reactive oxidized lipids leads to structural changes such as increased β-sheet conformation, which tends to result in amyloid-like structures and oligomerization, or unfolding and aggregation. Reaction with catalytic cysteines is often responsible for the loss of enzymatic activity in lipoxidized proteins, although inhibition may also occur through conformational changes at more distant sites affecting substrate binding or regulation. On the other hand, a few proteins are activated by lipoxidation-induced oligomerization or interactions, leading to increased downstream signalling. At the cellular level, it is clear that some proteins are much more susceptible to lipoxidation than others. ALEs affect cell metabolism, protein-protein interactions, protein turnover via the proteasome, and cell viability. Evidence is building that they play roles in both physiological and pathological situations, and inhibiting ALE formation can have beneficial effects.

Thermal Stability of Beef Myoglobin is Compromised by Reactive Lipid Oxidation Products

ObjectivesBrown color of cooked meat is the result of heat-induced denaturation of myoglobin (Mb). The denaturation temperature of Mb is governed by its redox state in raw meat; metmyoglobin (MMb) undergoes denaturation at a lower temperature than oxymyoglobin (OMb) and deoxymyoglobin (DMb). The secondary products of lipid oxidation, such as the 4-hydroxy-2-nonenal (HNE), compromise Mb redox stability and can thus impact cooked meat color. While previous investigations extensively studied lipid oxidation-induced Mb redox instability, studies are yet to be undertaken to examine the relationship between lipid oxidation and Mb thermal stability. Therefore, the objective of the present study was to investigate the direct influence of lipid oxidation (using HNE as a model aldehyde) on thermal stability of beef Mb at typical meat conditions.Materials and MethodsBeef Mb was purified, and OMb was incubated with HNE (0.15 mM Mb + 1.0 mM HNE) at pH 5.6 and 4°C for 21 d. The controls consisted of OMb plus a volume of ethanol used to deliver HNE to treatments. The samples were scanned spectrophotometrically from 650 to 500 nm on d 0, 7, 14, and 21, and MMb formation was calculated. The Mb samples were removed on d 0, 7, 14, and 21, and were digested with trypsin. The tryptic peptides were analyzed using liquid chromatography tandem-mass spectrometry (LC-MS/MS) for detecting HNE adduction sites. The thermal stability of Mb in the presence of HNE was assessed on d 0, 7, 14, and 21 by determining the percentage myoglobin denaturation (PMD) at 71°C in a water bath for 10 min. The experiment was replicated three times (n = 3). The effects of HNE on Mb redox and thermal stabilities during incubation were evaluated using the mixed procedure of SAS. The differences among means were detected at the 5% level using the least significant difference (LSD) test.ResultsWhile MMb formation increased (P < 0.05) during the storage in both control and HNE-treated samples, the oxidation was higher (P < 0.05) in HNE-treated samples. The PMD values increased (P < 0.05) in both treatments during the storage, and the HNE-treated samples exhibited greater (P < 0.05) PMD than the controls throughout the storage. Additionally, the PMD difference between HNE-treated and control samples increased over time. The LC-MS/MS analyses indicated that the number of histidines adducted by HNE increased with storage. HNE adducted four histidines (positions 24, 36, 93, and 152) on d 7, whereas five (positions 24, 36, 64, 93, and 152) and six (positions 24, 36, 64, 93, 113, and 152) residues were adducted on d 14 and 21, respectively.ConclusionThe mass spectrometric data indicated that thermal stability of beef Mb was compromised by reactive lipid oxidation products. The HNE adduction at the distal histidine (position 64), which is critical to heme stability, observed on d 14 and 21 as well as the increased number of histidines adducted by HNE on d 14 and 21 could be the possible reasons for the increased PMD on these time points. The adduction of HNE to histidines can alter the heme protein’s tertiary structure and thus exposes the heme to oxidation, thereby accelerating the formation of MMb, which is more susceptible to thermal denaturation than the ferrous Mb redox forms.

Thermal Stability of Beef Myoglobin is Compromised by Reactive Lipid Oxidation Products

Thermal Stability of Beef Myoglobin is Compromised by Reactive Lipid Oxidation Products

Prooxidant capacity of thermoxidised plant oils

The prooxidant capacity of rapeseed, sunflower, soybean, and olive oil was determined before and after heating at a temperature of 180°C for 2, 4, and 6 hours. It was quantified as losses of α-tocopherol caused by the studied oils during 24-h incubation of their acetone–methanol solutions with addition of α-tocopherol at 30°C, whereas the decrease in α-tocopherol concentration was studied as a decrease in antioxidant capacity determined by the spectrophotometric DPPH (2,2-diphenyl-1-picrylhydrazyl) method. During heating of all the studied plant oils, the prooxidant capacity grew due to the formation of reactive lipid oxidation products, but, except the sunflower oil, it did not depend on the time of heating – after the initiatory increase, the prooxidant capacity typically remained approximately constant or decreased. The prooxidant capacity of the heated oils ranged from 58 mg to 360 mg α-tocopherol/kg and decreased in the order soybean oil > rapeseed oil > olive oil ≈ sunflower oil. It did not correlate with the content of polymerised triacylglycerols (except the sunflower oil) and was generally higher than the residual content of α-tocopherol.

OP10 - Phospholipid oxidation of the skin and its role in stress response, autophagy and senescence

Oxidation of lipids and proteins is not only a manifestation of aged skin but also potentially causative for age-related aesthetic decline and pathologic damage. Extrinsic oxidative stress, like UV radiation promotes the accumulation of reactive lipid oxidation products. To study which oxidation products are generated by UV other age promoting stressors and in replicative senescence of keratinocytes, fibroblasts and melanocytes, we performed lipidomic analysis of oxidized and non-oxidized phospholipids (PL) using a HPLC-tandem-MS method accompanied by transcriptomic and proteomic profiling. With that approach we could quantify several hundred oxidized lipid species in KC after acute physiological UVA stress, exposure to promoters of cellular senescence like Paraquat over replicative senescence and in aged skin. Oxidized phospholipids activate the antioxidant response system in the various compartments of the skin, they regulate macroautophagy and they fine tune the innate immune system, and they are candidate molecules to be comprised in the senescence associated secretory phenotype of sencescent skin cells.

Redox proteomics: from protein modifications to cellular dysfunction and disease

1. Oxidatively Modified Proteins and Proteomic Technologies. 1.1 Chemical Modification of Proteins by Reactive Oxygen Species (E. Stadtman & R. Levine). 1.2 The Chemistry of Protein Modifications Elicited By Nitric Oxide and Related Nitrogen Oxides (D. Thomas, et al.). 1.3 Mass Spectrometry Approaches for the Molecular Characterization of Oxidatively/Nitrosatively Modified Proteins (A. Scaloni). 1.4 Thiol-disulfide Oxidoreduction of Protein Cysteines: Old Methods Revisted for Proteomics (V. Bonetto & P. Ghezzi). 1.5 Carbonylated Proteins and their Implication in Physiology and Pathology (R. Levine & E. Stadtman). 1.6 S-Nitrosation of Cysteine thiols as a Redox Signal (Y. Zhang & N. Hogg). 1.7 Detection of Glycated and GlycoOxidated Proteins (A. Lapolla, et al.). 1.8 MudPIT (Multidimensaional Protein Identification Technology) for Identification of Post-translational Protein Modifications in Complex Biological Mixtures (S. Thomas, et al.). 1.9 Use of a Proteomic Technique to Identify Oxidant-Sensitive Thiol Proteins in Cultured Cells (M. Hampton, et al.). 1.10 ICAT (Isotope-Code Affinity Tag) Approach to Redox Proteomics: Identification and Quantification of Oxidant-Sensitive Protein Thiols (M. Sethuraman, et al.). 1.11 Quantitative Determination of Free and Protein-Associated 3-nitrotyrosine and S-nitrosothiols in the Circulation by Mass Spectrometry and Other Methodologies: A Critical Review and Discussion from the Analytical and Review Point of View (D. Tsikas). 2. Cellular Aspects of Protein Oxidation. 2.1 The Covalent Advantage: A New Paradigm for Cell Signaling Mediated by Thiol Reactive Lipid Oxidation Products (D. Dickinson, et al.). 2.2 Early Molecular Events During Response to Oxidative Stress in Human Cells by Differential Proteomics (G. Tell). 2.3 Oxidative Damage to Proteins: Structural Modifications and Consequences in Cell Function (E. Cabiscol & J. Ros). 2.4 Oxidative Damage and Cellular Senescence: Lessons from Bacteria and Yeast (T. Nystrom). 3. Redox Proteomic Analysis in Human Diseases. 3.1 Proteins as Sensitive Biomarkers of Human Conditions Associated with Oxidative Stress (I. Dalle-Donne, et al.). 3.2 Degradation and Accumulation of Oxidized Proteins in Age-Related Diseases (P. Voss & T. Grune). 3.3 Redox Proteomics: A New Approach to Investgate Oxidative Stress in Alzheimer's Diseases (D. Butterfield, et al.). 3.4 Oxidized Proteins in Cardiac Ischemia-Reperfusion (J. Brennan & P. Eaton). 3.5 Proteome Anaylsis of Oxidative Stress: Glutathionyl Hemoglobin in Diabetic and Uremic Patients (T. Niwa). 3.6 Glyco-Oxidative Biochemistry in Diabetic Renal Injury (T. Miyata). 3.7 Quantitative Screnning of Protein Glycation, Oxidation, and Nitration Adducts by LC-MS/MS: Protein Damage in Diabetes, Uremia, Cirrhosis, and Alzheimer's Disease (P. Thornalley). 3.8 Protein Targets and Functional Consequences of Tyrosine Nitration in Vascular Disease (L. Baker, et al.). 3.9 Oxidation of Artery Wall Proteins by Myeloperoxidase: A proteomics Approach (T. Vaisar & J. Heinecke). 3.10 Oxidative Stress and Protein Oxidation in Pre-Eclampsia (M. Raijmakers, et al.). 3.11 The Involvement of Oxidants in the Etiology of Chronic Airway Diseases: Proteomic Approaches to Identify Redox Processes in Epithelial Cell Signal and Inflammation (A. van der Vliet, et al.). 3.12 Sequestering Agents of Intermediate Reactive Aldehydes as Inhibitors of Advanced Lipoxidation End-Products (ALEs) (M. Carini, et al.).

Mechanism of Suppression of Nrf2 Under Hypoxia

Lipid oxidation in foods is initiated by free radical and/or singlet oxygen mechanisms which generate a series of autocatalytic free radical reactions. These autoxidation reactions lead to the breakdown of lipid and to the formation of a wide array of oxidation products. The nature and proportion of these products can vary widely between foods and depend on the composition of the food as well as numerous environmental factors. The toxicological significance of lipid oxidation in foods is complicated by interactions of secondary lipid oxidation products with other food components. These interactions could either form complexes that limit the bioavailability of lipid breakdown products or can lead to the formation of toxic products derived from non-lipid sources. A lack of gross pathological consequences has generally been observed in animals fed oxidized fats. On the other hand, secondary products of lipid autoxidation can be absorbed and may cause an increase in oxidative stress and deleterious changes in lipoprotein and platelet metabolism. The presence of reactive lipid oxidation products in foods needs more systematic research in terms of complexities of food component interactions and the metabolic processing of these compounds.

Lipid Oxidation and Cardiovascular Disease: Introduction to a Review Series

A therosclerosis and the associated adverse complications of cardiovascular disease are major causes of morbidity and mortality in people living a Western lifestyle. A role for excess cholesterol in the pathophysiology of atherosclerosis is clear. However, additional mechanisms driving the relevant pathological changes in a chronic disease such as atherosclerosis are those that constitute the acute inflammatory response.1 The essential elements of a physiological, and regulated, inflammatory response starts with stimulated endothelium,2 displaying adhesive molecules for circulating white blood cells. This is accompanied by localized production of cell type–specific agonists for adherent monocytes, neutrophils, or lymphocytes by the activated endothelium.3 These agonists then activate the migratory instruction set of adherent or rolling cells positioned to receive both adhesion- and agonist-related stimuli from activated vascular endothelial cells.4 Lipid oxidation products formed by virtually every vascular cell type participate in orchestrating these processes. We also have recently come to appreciate that the inflammatory process is actively limited by activation of a resolution phase, often via generation of structurally specific oxidized lipids whose function is to orchestrate resolution of inflammation.5 The overall goal of this review series is to place recent observations and insights in rapidly developing areas of lipid oxidation into the framework of cardiovascular disease. Enzymatic and free radical oxidation have prominent roles in cardiovascular disease through their oxidative modification of existing molecules. Lipids are primary targets of this modification because they are the primary repository of oxidizable olefinic or double bonds. Oxidized lipids are best understood as oxygenated arachidonic acid products, the prostaglandins, leukotrienes, and thromboxane A2, that have diverse and potent effects throughout the inflammatory and reparative responses. These oxidized lipid mediators are …

Methods for imaging and detecting modification of proteins by reactive lipid species

Products of lipid peroxidation are generated in a wide range of pathologies associated with oxidative stress and inflammation. Many oxidized lipids contain reactive functional groups that can modify proteins, change their structure and function, and affect cell signaling. However, intracellular localization and protein adducts of reactive lipids have been difficult to detect, and the methods of detection rely largely on antibodies raised against specific lipid–protein adducts. As an alternative approach to monitoring oxidized lipids in cultured cells, we have tagged the lipid peroxidation substrate arachidonic acid and an electrophilic lipid, 15-deoxy-Δ 12,14-prostaglandin-J 2 (15d-PGJ 2), with either biotin or the fluorophore BODIPY. Tagged arachidonic acid can be used in combination with conditions of oxidant stress or inflammation to assess the subcellular localization and protein modification by oxidized lipids generated in situ. Furthermore, we show that reactive lipid oxidation products such as 15d-PGJ 2 can also be labeled and used in fluorescence and Western blotting applications. This article describes the synthesis, purification, and selected application of these tagged lipids in vitro.