Peroxidation Of Polyunsaturated Fatty Acids Research Articles

Perhydroxyl Radical (HO2^(•)) as Inducer of the Isoprostane Lipid Peroxidation in Mitochondria

The nonenzymatic isoprostane pathway of lipid peroxidation of polyunsaturated fatty acids results in formation of products, termed isoprostanes, which have very large positional and stereo isomerism, possess various biological activities, produce adducts with proteins, and thus contribute to pathogeneses of the age-dependent diseases. However, it was unclear what mechanism drives this type of lipid autoxidation, and why the products have very large isomerism. We propose a mechanism when perhydroxyl radicals (HO2^(•)) react with polyunsaturated fatty acids in the hydrophobic milieu of membranes. In the membrane HO2^(•) initiates a chain of reactions with formation first H2O2, which undergoes homolytic fission producing two ^(•)OH radicals, thus very rapidly abstracting three H atoms from a polyunsaturated fatty acid. As a result, the HO2^(•) molecule is converted to two molecules of water, and the molecule of a polyunsaturated fatty acid loses two double bonds, becomes highly unstable and undergoes peroxidation and random intramolecular re-arrangements causing a very large isomerism of the final products. The extremely high reactivity of ^(•)HО2 with polyunsaturated fatty acids is the cause of very subtle and slow accumulation of damages in the membrane and membrane associated proteins, even though the concentration of ^(•)HО2 relative to superoxide radical may be very low.

Modification of α-synuclein by lipid peroxidation products derived from polyunsaturated fatty acids promotes toxic oligomerization: its relevance to Parkinson disease.

Recently, toxic α-synuclein oligomer, which can mediate cell-to-cell propagation is suggested to cause sporadic Parkinson disease. α-Synuclein interacts with membrane lipids especially polyunsaturated fatty acids to stabilize its three-dementional structure. Peroxidation of polyunsaturated fatty acids may reduce their affinity to α-synuclein and peroxidation byproducts might modify α-synuclein. 4-Hydroxy-2-nonenal derived from n-6 polyunsaturated fatty acids was reported to modify α-synuclein to produce a toxic oligomer. Moreover, the accumulation of 4-hydroxy-2-nonenal, which could induce oligomeriztion of α-synuclein, was found in parkinsonian brains. Docosahexaenoic acid, an n-3 polyunsaturated fatty acids abundant in the neuronal membrane, was also found to enhance α-synuclein oligomerization; however, the precise details of the chemical reaction involved are unclear. Propanoylated lysine, a specific indicator of docosahexaenoic acid oxidation, was increased in neuronal differentiated human neuroblastoma SH-SY5Y cells overexpressing α-synuclein. α-Synuclein might be modified by the peroxidation products and then, is degraded by the autophagy-lysosome system. In addition, in the cells overexpressing α-synuclein, the mitochondrial electrone transfer chain was found to be inhibited. Accumulation of abnormal α-synuclein modified by lipid radicals derived from polyunsaturated fatty acids may be not only an indicator of brain oxidative stress but also causative of neurodegeneration such as Parkinson disease by impairing mitochondrial function.

Chronic vitamin E deficiency impairs cognitive function in adult zebrafish via dysregulation of brain lipids and energy metabolism

Zebrafish (Danio rerio) are a recognized model for studying the pathogenesis of cognitive deficits and the mechanisms underlying behavioral impairments, including the consequences of increased oxidative stress within the brain. The lipophilic antioxidant vitamin E (α-tocopherol; VitE) has an established role in neurological health and cognitive function, but the biological rationale for this action remains unknown. In the present study, we investigated behavioral perturbations due to chronic VitE deficiency in adult zebrafish fed from 45 days to 18-months of age diets that were either VitE-deficient (E–) or VitE-sufficient (E+). We hypothesized that E– zebrafish would display cognitive impairments associated with elevated lipid peroxidation and metabolic disruptions in the brain. Quantified VitE levels at 18-months in E– brains (5.7 ± 0.1 nmol/g tissue) were ~20-times lower than in E+ (122.8 ± 1.1; n = 10/group). Using assays of both associative (avoidance conditioning) and non-associative (habituation) learning, we found E– vs E+ fish were learning impaired. These functional deficits occurred concomitantly with the following observations in adult E– brains: decreased concentrations of and increased peroxidation of polyunsaturated fatty acids (especially docosahexaenoic acid, DHA), altered brain phospholipid and lysophospholipid composition, as well as perturbed energy (glucose/ketone), phosphatidylcholine and choline/methyl-donor metabolism. Collectively, these data suggest that chronic VitE deficiency leads to neurological dysfunction through multiple mechanisms that become dysregulated secondary to VitE deficiency. Apparently, the E– animals alter their metabolism to compensate for the VitE deficiency, but these compensatory mechanisms are insufficient to maintain cognitive function.

Development of 4-hydrazinyl-7-nitrobenzofurazan as a fluorogenic probe for detecting malondialdehyde in biological samples

Malondialdehyde (MDA) is a critical product of polyunsaturated fatty acid peroxidation and represents a common biomarker of oxidative stress. In this study, we have developed a fluorogenic MDA probe, 4-hydrazinyl-7-nitrobenzofurazan (NBDH). The reaction between NBDH and MDA in acidic conditions yields a highly fluorescent product, 4,5-dihydro-1H-pyrazole derivative (NBD-P1). The MDA detection range of our probe NBDH falls between 0.1 and 20μM, and the corresponding detection limit amounts to 7.2nM (3δ). NBDH also demonstrates a high selectivity toward MDA over other aldehydes and biologically relevant molecules, and proves to be an excellent MDA assay in biological fluids.

Fatty acid-based lipidomics and membrane remodeling induced by apoE3 and apoE4 in human neuroblastoma cells

Apolipoprotein E (apoE) is a major lipid carrier of the lipoprotein transport system that plays critical roles in various pathologies. Human apoE has three common isoforms, the apoE4 being associated with Alzheimer's disease. This is the first study in the literature investigating the effects of apoE (apoE3 and apoE4 isoforms) on membrane fatty acid profile in neuroblastoma SK-N-SH cells. Fatty acid analyses were carried out by gas chromatography of the corresponding methyl esters (FAME). We observed the occurrence of membrane fatty acid remodeling in the presence of each of the two apoE isoforms. ApoE3 increased the membrane level of stearic acid and dihomo-gamma-linolenic acid (DGLA), whereas apoE4 had opposite effects. Both apoE3 and apoE4 increased saturated and monounsaturated fatty acids (SFA and MUFA), omega-6/omega-3 ratio and decreased total polyunsaturated fatty acid (PUFA) amount, but with various intensities. Moreover, both apoE isoforms decreased membrane homeostasis indexes such as PUFA balance, unsaturation index and peroxidation index. Our results highlight membrane property changes connected to the apoE isoforms suggesting membrane lipidomics to be inserted in further model studies of apolipoproteins in health and disease.

Time courses of post-injury mitochondrial oxidative damage and respiratory dysfunction and neuronal cytoskeletal degradation in a rat model of focal traumatic brain injury

Traumatic brain injury (TBI) results in rapid reactive oxygen species (ROS) production and oxidative damage to essential brain cellular components leading to neuronal dysfunction and cell death. It is increasingly appreciated that a major player in TBI-induced oxidative damage is the reactive nitrogen species (RNS) peroxynitrite (PN) which is produced in large part in injured brain mitochondria. Once formed, PN decomposes into highly reactive free radicals that trigger membrane lipid peroxidation (LP) of polyunsaturated fatty acids (e.g. arachidonic acid) and protein nitration (3-nitrotyrosine, 3-NT) in mitochondria and other cellular membranes causing various functional impairments to mitochondrial oxidative phosphorylation and calcium (Ca2+) buffering capacity. The LP also results in the formation of neurotoxic reactive aldehyde byproducts including 4-hydroxynonenal (4-HNE) and propenal (acrolein) which exacerbates ROS/RNS production and oxidative protein damage in the injured brain. Ultimately, this results in intracellular Ca2+ overload that activates proteolytic degradation of α-spectrin, a neuronal cytoskeletal protein. Therefore, the aim of this study was to establish the temporal evolution of mitochondrial dysfunction, oxidative damage and cytoskeletal degradation in the brain following a severe controlled cortical impact (CCI) TBI in young male adult rats. In mitochondria isolated from an 8 mm diameter cortical punch including the 5 mm wide impact site and their respiratory function studied ex vivo, we observed an initial decrease in complex I and II mitochondrial bioenergetics within 3 h (h). For complex I bioenergetics, this partially recovered by 12–16 h, whereas for complex II respiration the recovery was complete by 12 h. During the first 24 h, there was no evidence of an injury-induced increase in LP or protein nitration in mitochondrial or cellular homogenates. However, beginning at 24 h, there was a gradual secondary decline in complex I and II respiration that peaked at 72 h. post-TBI that coincided with progressive peroxidation of mitochondrial and cellular lipids, protein nitration and protein modification by 4-HNE and acrolein. The oxidative damage and respiratory failure paralleled an increase in Ca2+-induced proteolytic degradation of the neuronal cytoskeletal protein α-spectrin indicating a failure of intracellular Ca2+ homeostasis. These findings of a surprisingly delayed peak in secondary injury, suggest that the therapeutic window and needed treatment duration for certain antioxidant treatment strategies following CCI-TBI in rodents may be longer than previously believed.

Crystal Structure of the Chloroplastic Oxoene Reductase ceQORH from Arabidopsis thaliana.

Enzymatic and non-enzymatic peroxidation of polyunsaturated fatty acids give rise to accumulation of aldehydes, ketones, and α,β-unsaturated carbonyls of various lengths, known as oxylipins. Oxylipins with α,β-unsaturated carbonyls are reactive electrophile species and are toxic. Cells have evolved several mechanisms to scavenge reactive electrophile oxylipins and decrease their reactivity such as by coupling with glutathione, or by reduction using NAD(P)H-dependent reductases and dehydrogenases of various substrate specificities. Plant cell chloroplasts produce reactive electrophile oxylipins named γ-ketols downstream of enzymatic lipid peroxidation. The chloroplast envelope quinone oxidoreductase homolog (ceQORH) from Arabidopsis thaliana was previously shown to reduce the reactive double bond of γ-ketols. In marked difference with its cytosolic homolog alkenal reductase (AtAER) that displays a high activity toward the ketodiene 13-oxo-9(Z),11(E)-octadecadienoic acid (13-KODE) and the ketotriene 13-oxo-9(Z), 11(E), 15(Z)-octadecatrienoic acid (13-KOTE), ceQORH binds, but does not reduce, 13-KODE and 13-KOTE. Crystal structures of apo-ceQORH and ceQORH bound to 13-KOTE or to NADP+ and 13-KOTE have been solved showing a large ligand binding site, also observed in the structure of the cytosolic alkenal/one reductase. Positioning of the α,β-unsaturated carbonyl of 13-KOTE in ceQORH-NADP+-13-KOTE, far away from the NADP+ nicotinamide ring, provides a rational for the absence of activity with the ketodienes and ketotrienes. ceQORH is a monomeric enzyme in solution whereas other enzymes from the quinone oxidoreductase family are stable dimers and a structural explanation of this difference is proposed. A possible in vivo role of ketodienes and ketotrienes binding to ceQORH is also discussed.

Short-time UVA exposure to human keratinocytes instigated polyunsaturated fatty acid without inducing lipid peroxidation

Short-term exposure to ultraviolet A (UVA) radiation can directly injure our skin through inflammatory response and indirectly through oxidative stress, triggering polyunsaturated fatty acid (PUFA) peroxidation in skin cell membrane and formation of DNA adduct, 8-hydroxy-2′-deoxyguanosine (8-OHdG). It is known that UVA exposure leads to photoaging, immunosuppression and skin cancer. However, the changes in PUFA and its oxidized metabolites, and cell cycle after short UVA exposure, are debatable. In this study, human keratinocytes (HaCaT) were exposed to low dose (5 J/cm2) and high dose (20 J/cm2) of UVA and assessed immediately, 8 h, 12 h, and 24 h post-treatment. Both doses showed a transient suppression in S-phase after 8 h of UVA exposure, and G2/M phase arrest after 12-h UVA exposure in the cell cycle but subsequently returned to normal cycle. Also, no observable DNA damage took place, where 8-OHdG levels were below par after 24-h UVA exposure. A dose of 20 J/cm2 UVA stimulated significant amount of arachidonic acid, n-3 docosapentaenoic acid, and docosahexaenoic acid (DHA) but lowered adrenic acid and eicospentaenoic acid after 24-h exposure. Among the 43 oxidized PUFA products determined, enzyme-dependent oxidized PUFAs, namely, 14-hydroxy-DHA (HDoHE) level reduced, and 8- and 13-HDoHE levels elevated significantly in a linear trend with post-treatment time. Out of the nonenzymatic oxidized PUFAs, a significant linear trend with post-treatment time was shown on the reduction of 5-F2t-Isoprostane (IsoP), 15-F2t-IsoP, Isofurans, 5-F3t-IsoP, Neurofurans, and 20-HDoHE. Our observations indicate oxidative stress through short UVA exposure on human keratinocytes did not have detrimental consequences.

Potential applications of lipid peroxidation products – F4-neuroprostanes, F3-neuroprostanesn-6 DPA, F2-dihomo-isoprostanes and F2-isoprostanes ‐ in the evaluation of the allograft function in renal transplantation

F4-neuroprostanes, F3-neuroprostanesn-6 DPA, and F2-dihomo-isoprostanes, metabolites of non-enzymatic lipid peroxidation of polyunsaturated fatty acids [docosahexaenoic acid, n-6 docosapentanoic acid, and adrenic acid respectively], have become important biomarkers for oxidative stress in several diseases like epilepsy and alzheimer. These biomarkers and the 15-F2t-isoprostane (also known as 8-iso-PGF2α), a F2-isoprostane isomer measured as reference oxidative marker at systemic level, were analyzed by UHPLC-QqQ-MS/MS in the urine of 60 renal recipients from cadaveric donors of the Nephrology Unit of the University Hospital Virgen de la Arrixaca, at six different times during the first six months after renal transplantation, and were compared with a control group of 60 healthy subjects from the same hospital. A total of 11 metabolites were analyzed and different patterns were observed. A tendency to decrease was observed in three metabolites (4-epi−4-F3t- NeuroPn-6 DPA, ent−7(RS)−7-F2t-dihomo-IsoP, and ent−7(S)−7-F2t-dihomo-IsoP) and in our reference oxidative marker (15-F2t-IsoP) when kidney function improved and the excretion of urine proteins decreased. These results suggest that these three biomarkers of oxidative stress could be useful to assess renal function in the postransplant phase. Unfortunately, little is known about this kind of biomarker in this cohort of patients, so further investigation would be required in the clinical field to clarify the relationship between oxidative stress and the graft function, as well as the usefulness of these biomarkers as rejection markers.

Non-enzymatic oxidized metabolite of DHA, 4(RS)-4-F4t-neuroprostane protects the heart against reperfusion injury

Acute myocardial infarction leads to an increase in oxidative stress and lipid peroxidation. 4(RS)-4-F4t-Neuroprostane (4-F4t-NeuroP) is a mediator produced by non-enzymatic free radical peroxidation of the cardioprotective polyunsaturated fatty acid, docosahexaenoic acid (DHA). In this study, we investigated whether intra-cardiac delivery of 4-F4t-NeuroP (0.03mg/kg) prior to occlusion (ischemia) prevents and protects rat myocardium from reperfusion damages.Using a rat model of ischemic-reperfusion (I/R), we showed that intra-cardiac infusion of 4-F4t-NeuroP significantly decreased infarct size following reperfusion (−27%) and also reduced ventricular arrhythmia score considerably during reperfusion (−41%). Most notably, 4-F4t-NeuroP decreased ventricular tachycardia and post-reperfusion lengthening of QT interval. The evaluation of the mitochondrial homeostasis indicates a limitation of mitochondrial swelling in response to Ca2+ by decreasing the mitochondrial permeability transition pore opening and increasing mitochondria membrane potential. On the other hand, mitochondrial respiration measured by oxygraphy, and mitochondrial ROS production measured with MitoSox red® were unchanged. We found decreased cytochrome c release and caspase 3 activity, indicating that 4-F4t-NeuroP prevented reperfusion damages and reduced apoptosis. In conclusion, 4-F4t-NeuroP derived from DHA was able to protect I/R cardiac injuries by regulating the mitochondrial homeostasis.

Assessment of Isoprostanes in Human Plasma: Technical Considerations and the Use of Mass Spectrometry.

Oxygenated lipid mediators released from non-enzymatic peroxidation of polyunsaturated fatty acids (PUFA) are known to have functional roles in humans. Notably, among these lipid mediators, isoprostanes molecules are robust biomarkers of oxidative stress but those from n-3 PUFA are also bioactive molecules. In order to identify and assess the isoprostanes, the use of mass spectrometry (MS) for analysis is preferable and has been used for over two decades. Gas chromatography (GC) is commonly coupled to the MS to separate the derivatized isoprostanes of interest in biological samples. In order to increase the accuracy of the analytical performance, GC-MS/MS was also applied. Lately, MS or MS/MS has been coupled with high-performance liquid chromatography to assess multiple isoprostane molecules in a single biological sample without derivatization process. However, there are limitations for the use of LC-MS/MS in the measurement of plasma isoprostanes, which will be discussed in this review.

Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis

Ferroptosis is form of regulated nonapoptotic cell death that is involved in diverse disease contexts. Small molecules that inhibit glutathione peroxidase 4 (GPX4), a phospholipid peroxidase, cause lethal accumulation of lipid peroxides and induce ferroptotic cell death. Although ferroptosis has been suggested to involve accumulation of reactive oxygen species (ROS) in lipid environments, the mediators and substrates of ROS generation and the pharmacological mechanism of GPX4 inhibition that generates ROS in lipid environments are unknown. We report here the mechanism of lipid peroxidation during ferroptosis, which involves phosphorylase kinase G2 (PHKG2) regulation of iron availability to lipoxygenase enzymes, which in turn drive ferroptosis through peroxidation of polyunsaturated fatty acids (PUFAs) at the bis-allylic position; indeed, pretreating cells with PUFAs containing the heavy hydrogen isotope deuterium at the site of peroxidation (D-PUFA) prevented PUFA oxidation and blocked ferroptosis. We further found that ferroptosis inducers inhibit GPX4 by covalently targeting the active site selenocysteine, leading to accumulation of PUFA hydroperoxides. In summary, we found that PUFA oxidation by lipoxygenases via a PHKG2-dependent iron pool is necessary for ferroptosis and that the covalent inhibition of the catalytic selenocysteine in Gpx4 prevents elimination of PUFA hydroperoxides; these findings suggest new strategies for controlling ferroptosis in diverse contexts.

Inhibition of β-oxidation is not a valid therapeutic tool for reducing oxidative stress in conditions of neurodegeneration

According to recent reports, systemic treatment of rats with methylpalmoxirate (carnitine palmitoyltransferase-1 inhibitor) decreased peroxidation of polyunsaturated fatty acids in brain tissue. This was taken as evidence of mitochondrial β-oxidation in brain, thereby contradicting long-standing paradigms of cerebral metabolism, which claim that β-oxidation of activated fatty acids has minor importance for brain energy homeostasis. We addressed this controversy. Our experiments are the first direct experimental analysis of this question. We fueled isolated brain mitochondria or rat brain astrocytes with octanoic acid, but octanoic acid does not enhance formation of reactive oxygen species, neither in isolated brain mitochondria nor in astrocytes, even at limited hydrogen delivery to mitochondria. Thus, octanoic acid or l-octanoylcarnitine does not stimulate H2O2 release from brain mitochondria fueled with malate, in contrast to liver mitochondria (2.25-fold rise). This does obviously not support the possible occurrence of β-oxidation of the fatty acid octanoate in the brain. We conclude that a proposed inhibition of β-oxidation does not seem to be a helpful strategy for therapies aiming at lowering oxidative stress in cerebral tissue. This question is important, since oxidative stress is the cause of neurodegeneration in numerous neurodegenerative or inflammatory disease situations.

Abstract 1713: Macrophage-induced bystander effect activates Wnt/β-catenin signaling and induces cellular dedifferentiation

Abstract Cancer stem cells (CSCs) in colorectal cancer (CRC) help maintain tumor heterogeneity, promote tumor growth, invasion, and metastasis, and produce resistance to therapeutic agents. The origin of colorectal CSCs, however, is unclear. Our recent studies showed that a macrophage-induced bystander effect (MIBE) induced gene expression of stem/progenitor cell markers including Ly6A/E and Dclk1 in murine colonic epithelial cells, implying that MIBE helps induce colorectal CSCs. In current studies we show that commensal-triggered MIBE activates Wnt/β-catenin signaling and induces dedifferentiation of colonic epithelial cells during colorectal carcinogenesis, thus contributing to the origin of colorectal CSCs. Exposure of murine primary epithelial cells (YAMC) to commensal-polarized macrophages induced phosphorylated Gsk3β (Ser9), active β-catenin, and Tcf4, suggesting activation of Wnt/β-catenin signaling by MIBE. In vivo, active β-catenin was evident in both stromal and epithelial cells for colon biopsies from E. faecalis-colonized Il10−/− mice compared to sham-colonized mice. In addition, we showed that 4-hydroxynonenal (4-HNE), a byproduct of lipid peroxidation of ω6 polyunsaturated fatty acids, and tumor necrosis factor α (TNFα) mediated activation of Wnt/β-catenin signaling. Epigenetic analysis revealed that MIBE activated Wnt/β-catenin signaling through DNA methylation in the CpG islands of secreted frizzled-related protein 2 (Sfrp2), a soluble modulator of Wnt. Next, we showed that exposure of YAMC cells to 4-HNE and TNFα induced expression of c-Myc, Klf4, Oct4, and Sox2―factors essential for pluripotent stem cells. Immunofluorescent staining of colon biopsies from E. faecalis-colonized Il10−/− mice showed increased expression of these factors in colonic epithelial cells, suggesting dedifferentiation induced by MIBE. This was confirmed by qRT-PCR and Western blots that showed increased expression of Dclk1 and CD44, two markers for colorectal CSCs. Furthermore, increased expression of DCLK1 was noted in human tubular adenomas and invasive CRCs compared to hyperplastic polyps. Finally, inhibition of β-catenin/TCF4 using FH535, and by silencing CTNNB1, decreased DCLK1 expression in HCT116 human colon cancer cells, supporting the notion that Wnt/β-catenin signaling induces cellular dedifferentiation leading to colorectal CSCs. In summary, these results demonstrate that commensal-polarized macrophages activate Wnt/β-catenin signaling and induce dedifferentiation of colonic epithelial cells through 4-HNE and TNFα-mediated bystander effects. These findings provide new evidence for the origin of CSCs in colorectal carcinogenesis and should lead to novel strategies for CRC prevention and therapy. Citation Format: Xingmin Wang, Yonghong Yang, Mark M. Huycke. Macrophage-induced bystander effect activates Wnt/β-catenin signaling and induces cellular dedifferentiation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1713.

Effect of antioxidants on polyunsaturated fatty acids - review.

This paper aims to review the available literature data and provide an overview regarding the efficiency of antioxidants to prevent peroxidation of polyunsaturated fatty acids. Lipid peroxidation is a serious problem that often leads to a loss of shelf-life, reduced consumer acceptability, poorer functionality, lower nutritional value, and poorer safety. It represents an oxidative degradation of polyunsaturated fatty acids incorporated in cell membrane lipids or in lipoproteins, but also in vegetables and food oils rich in PUFA n-3. It is a complex process that leads to the production of numerous highly reactive metabolites with consequences for food preservation and for the development of various diseases. The targets of lipid oxidation are polyunsaturated fatty acids. Lipid peroxidation can proceed by means of two different reactions that lead to the formation of hydroperoxides as primary products. Hydroperoxides decompose rapidly to give many secondary products, such as lipid free radicals, which contribute to increased oxidation of other molecules, such as proteins, nucleic acids and other lipids. Lipid peroxidation is a major problem for the food industry, as well as for human health, since it is associated with many diseases. The use of antioxidants reduces oxidative damage.

Identification of 4-hydroxynonenal protein targets in rat, mouse and human liver microsomes by two-dimensional liquid chromatography/tandem mass spectrometry.

4-Hydroxynonenal (HNE), endogenously generated through peroxidation and breakdown of polyunsaturated fatty acids, has been linked to a number of adverse biological effects through carbonylation of essential biomolecules. Covalent binding of HNE to proteins can alter their structure and functions, causing cell damage as well as adverse immune responses. The liver plays a predominant role in metabolic transformations and hepatic proteins are often targeted by reactive metabolites. Rat, mouse and human liver microsomes were incubated with HNE, enzymatically digested, and subjected to strong cation-exchange peptide fractionation prior to liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis coupled to electrospray ionization quadrupole time-of-flight (QqTOF) mass spectrometry. HNE-modified peptides were detected by probability-driven peptide spectral matching and comparative analysis between treated and control samples, and confirmed based on accurate mass and high-resolution MS/MS spectra. A total of 99, 123 and 51 HNE-modified peptides were identified in rat, mouse and human liver microsomes related to 76, 103 and 44 target proteins, respectively. Eight proteins were found to be adducted by HNE in all three species, including ATP synthase, carbamoyl phosphate synthase, cytochrome P450 1A2, glutamate dehydrogenase 1, protein ERGIC-53, protein disulfide-isomerase, and voltage-dependent anion-selective channel protein 1. These proteins play crucial roles in cellular processes and their covalent modification could potentially alter their function and lead to cytotoxicity. An analytical approach was developed for the identification of in vitro HNE protein targets in rat, mouse and human liver microsomes using two-dimensional (2D) LC/MS/MS. This approach can be applied to study HNE modification of proteins in vitro and in vivo, providing insight into the toxicology of HNE protein adduction. Copyright © 2016 John Wiley & Sons, Ltd.

SIÇANLARIN KALP DOKULARINDA YAĞ ASİTLERİ DÜZEYLERİNİN I/R VE MELATONİN UYGULAMALARIYLA DEĞİŞİMLERİNİN İNCELENMESİ

Melatonin (N-acetyl-5-methoxytryptamine),the main secretory product of the pineal gland, is a free radical scavenger that has been found to protect against lipid peroxidation in many experimental models. In the present many study the effect of melatonin on lipid peroxidation of long chain polyunsaturated fatty acids located in rat. The most sensitive fatty acids in the heart were the monounsaturated eicosenoik acid (C20:1 n-6), polyunsaturated arachidonic acid (C20:4 n-6) docosapenthonoic acid (C22:5 n-3) and docosahexanoic acid (C22:6 n-3) which compared with the corresponding control values. I/R performed group showed remarkable differerences (p<0.05).In this study, myokardial-iscemia reperfusion is determined to cause increase or decreasa in different types of fatty acids.

Volatile emission in dry seeds as a way to probe chemical reactions during initial asymptomatic deterioration.

The nature and kinetics of reactions in dry seeds determines how long the seeds survive. We used gas chromatography to assay volatile organic compounds (VOCs) emitted from seeds of three unrelated species as a means to non-invasively probe chemical changes during very dry, dry, and humid storage (seeds were dried to 5.5, 33, and 75% relative humidity at room temperature). VOCs emitted from seeds stored in humid conditions reflected fermentation-type reactions, with methanol and ethanol being predominant in Lactuca sativa and Carum carvi, and acetaldehyde and acetone being predominant in Eruca vesicaria. Dried C. carvi seeds continued to emit fermentation-type products, although at slower rates than the seeds stored in humid conditions. In contrast, drying caused a switch in VOC emission in L. sativa and E. vesicaria seeds towards higher emission of pentane and hexanal, molecules considered to be byproducts from the peroxidation of polyunsaturated fatty acids. Longevity correlated best with the rate of fermentation-type reactions and appeared unrelated to the rate of lipid peroxidation. Emission of VOCs decreased when seed species were mixed together, indicating that seeds adsorbed VOCs. Adsorption of VOCs did not appear to damage seeds, as longevity was not affected in seed mixtures. Collectively, the study shows similarity among species in the types of reactions that occur in dry seeds, but high diversity in the substrates, and hence the byproducts, of the reactions. Moreover, the study suggests that the most abundant VOCs arise from degradation of storage reserves within seed cells, and that these reactions and their byproducts are not, in themselves, damaging.

Malondialdehyde induces autophagy dysfunction and VEGF secretion in the retinal pigment epithelium in age-related macular degeneration

Age-related macular degeneration (AMD) is a major cause of blindness in developed countries and is closely related to oxidative stress, which leads to lipid peroxidation. Malondialdehyde (MDA) is a major byproduct of polyunsaturated fatty acid (PUFA) peroxidation. Increased levels of MDA have been reported in eyes of AMD patients. However, little is known about the direct relationship between MDA and AMD. Here we show the biological importance of MDA in AMD pathogenesis. We first confirmed that MDA levels were significantly increased in eyes of AMD patients. In ARPE-19 cells, a human retinal pigment epithelial cell line, MDA treatment induced vascular endothelial growth factor (VEGF) expression alternation, cell junction disruption, and autophagy dysfunction that was also observed in eyes of AMD patients. The MDA-induced VEGF increase was inhibited by autophagy–lysosomal inhibitors. Intravitreal MDA injection in mice increased laser-induced choroidal neovascularization (laser-CNV) volumes. In a mouse model fed a high-linoleic acid diet for 3 months, we found a significant increase in MDA levels, autophagic activity, and laser-CNV volumes. Our study revealed an important role of MDA, which acts not only as a marker but also as a causative factor of AMD pathogenesis-related autophagy dysfunction. Furthermore, higher dietary intake of linoleic acid promoted CNV progression in mice with increased MDA levels.

Volatile organic compounds discriminate between eosinophilic and neutrophilic inflammation in vitro

Inflammation associated oxidative stress leads to peroxidation of polyunsaturated fatty acids thereby generating volatile organic compounds (VOCs). The integrative analysis of the total amount of VOCs released by eosinophils and neutrophils in vitro enables the search for those compounds that discriminates between various inflammatory conditions.The approach comprises isolating eosinophils and neutrophils from 30 ml of blood of healthy non-smoking volunteers by gradient centrifugation, using lymphoprep. Eosinophils are separated from neutrophils by immunomagnetic cell separation using anti-CD16. Cells are activated with phorbol 12-myristate 13-acetate and VOCs from the headspace are collected at time 0′, 30′, 60′ and 90′ by introduction of ultra-pure nitrogen in the closed flasks at a flow rate of 200 ml min−1 during 10 min. The gases are trapped onto a sorption tube and analyzed by gas chromatography—time-of-flight—mass spectometry (GC-TOF-MS) in order to identify VOCs released in the headspace by activated neutrophils and eosinophils.Eosinophils and neutrophils were isolated from 26 healthy non-smoking volunteers. The average absolute number of eosinophils and neutrophils upon isolation was 3.5 × 106 and 19.4 × 106, respectively. The volatome in headspace consisted of 2116 compounds and those compounds present in at least 8% of the samples (1123 compounds) were used for further discriminant analysis. Discriminant analysis showed that two VOCs were able to distinguish between eosinophilic and neutrophilic cultures in the unactivated state with 100% correct classification of the entire data set and upon cross validation while five VOCs were able to discriminate between activated eosinophils and neutrophils with 96% correct classification in the original set and upon cross-validation.Analysis of VOCs seems to be a very promising approach in identifying eosinophilic and neutrophilic inflammation but it needs further development and in vivo confirmation.