Arachidonic Acid Oxidation Products Research Articles

Lipidomic changes in a novel sepsis outcome-based analysis reveals potent pro-inflammatory and pro-resolving signaling lipids.

The purpose of this study was to investigate changes in the lipidome of patients with sepsis to identify signaling lipids associated with poor outcomes that could be linked to future therapies. Adult patients with sepsis were enrolled within 24h of sepsis recognition. Patients meeting Sepsis-3 criteria were enrolled from the emergency department or intensive care unit and blood samples were obtained. Clinical data were collected and outcomes of rapid recovery, chronic critical illness (CCI), or early death were adjudicated by clinicians. Lipidomic analysis was performed on two platforms, the Sciex™ 5500 device to perform a lipidomic screen of 1450 lipid species and a targeted signaling lipid panel using liquid-chromatography tandem mass spectrometry. For the lipidomic screen, there were 274 patients with sepsis: 192 with rapid recovery, 47 with CCI, and 35 with early deaths. CCI and early death patients were grouped together for analysis. Fatty acid (FA) 12:0 was decreased in CCI/early death, whereas FA 17:0 and 20:1 were elevated in CCI/early death, compared to rapid recovery patients. For the signaling lipid panel analysis, there were 262 patients with sepsis: 189 with rapid recovery, 45 with CCI, and 28 with early death. Pro-inflammatory signaling lipids from ω-6 poly-unsaturated fatty acids (PUFAs), including 15-hydroxyeicosatetraenoic (HETE), 12-HETE, and 11-HETE (oxidation products of arachidonic acid [AA]) were elevated in CCI/early death patients compared to rapid recovery. The pro-resolving lipid mediator from ω-3 PUFAs, 14(S)-hydroxy docosahexaenoic acid (14S-HDHA), was also elevated in CCI/early death compared to rapid recovery. Signaling lipids of the AA pathway were elevated in poor-outcome patients with sepsis and may serve as targets for future therapies.

Identification of oxygenated triacylglycerols in pistachio nuts’ lipids by reverse-phase high-resolution-liquid chromatography-ESI mass spectrometry

Hydroxy- and peroxy-triacylglycerols are common products of lipid peroxidation formed during oil storage or heating or as enzymatic oxidation product of arachidonic acid as signaling molecules in mammals. In this study, oxygenated triacylglycerides (TAG) were identified in pistachio oil based on reverse phase(RP), high-performance liquid chromatography coupled with electrospray ionization and mass spectrometry (HPLC- ESI -MS). 20 novel lipid plant metabolites, classified based on their fragment spectra into a hydroxy (TAG-OH), an epoxy (TAG-O), and hydroperoxide (TAG-OOH) groups. We believe that this class of compounds has been for the first time observed as genuine secondary plant metabolites in a natural source in this case pistachio lipids of dietary relevance.

Multiple selected ion monitoring mode for sensitive imaging of eicosanoids in tissues using nanospray desorption electrospray ionization (nano-DESI) mass spectrometry

Nanospray desorption electrospray ionization mass spectrometry imaging (nano-DESI MSI) enables sensitive label-free mapping of multiple classes of molecules in biological samples. Previously, we demonstrated that combining nano-DESI MSI with selected ion monitoring (SIM) increases the sensitivity of the technique in a narrow m/z range. In this study, we use a series of adjacent multiple SIM (mSIM) windows to acquire nano-DESI MSI data with high sensitivity over a broader m/z range. We demonstrate that this approach substantially improves the quality of imaging data obtained for low-abundance eicosanoids, important lipid mediators derived from eicosapolyenoic acid products of arachidonic acid oxidation. Despite their importance as mediators of inflammation, asthma, fever, pain, hypertension, and stroke, eicosanoids have not been extensively investigated using MSI. This is mainly attributed to their low ionization efficiency and low, subnanomolar, concentrations in tissue, which make it difficult to detect these species in MSI experiments alongside abundant phospholipids. We demonstrate substantial enhancements in the signal-to-noise ratios (S/N) of eicosanoids using imaging in mSIM mode. We have also developed data processing and visualization tools to support this type of MSI data acquisition. S/N enhancements observed in mSIM mode are independent of the properties of analytes making this approach applicable to other low-abundance species in biological and environmental samples.

Arachidonic Acid in Follicular Fluid of PCOS Induces Oxidative Stress in a Human Ovarian Granulosa Tumor Cell Line (KGN) and Upregulates GDF15 Expression as a Response.

Polycystic ovarian ovary syndrome (PCOS) is the main cause of ovulatory infertility and a common reproductive endocrine disease of women in reproductive age. In addition, nearly half of PCOS patients are associated with obesity, and their total free fatty acids tend to increase. Arachidonic acid (AA) is a polyunsaturated fatty acid. Oxidation products of AA reacting with various enzymes[cyclooxygenases (COX), lipoxygenases (LOX), cytochrome P450s (CYP)] can change cellular mitochondrial distribution and calcium ion concentration, and increase reactive oxygen species (ROS) production. In this study, we analyzed the follicular fluid fatty acids and found higher levels of C20:4n6 (AA) in PCOS patients than in normal control subjects. Also, to determine whether AA induces oxidative stress (OS) in the human ovarian granulosa tumor cell line (KGN) and affects its function, we treated KGN cells with or without reduced glutathione (GSH) and then stimulated them with AA. The results showed that AA significantly reduced the total antioxidant capacity (TAC) and activity of antioxidant enzymes and increased the malondialdehyde (MDA), ROS and superoxide anion(O2-)levels in KGN cells. In addition, AA was also found to impair the secretory and mitochondrial functions of KGN cells and induce their apoptosis. We further investigated the downstream genes affected by AA in KGN cells and its mechanism of action. We found that AA upregulated the expression of growth differentiation factor 15 (GDF15), which had a protective effect on inflammation and tissue damage. Therefore, we investigated whether AA-induced OS in KGN cells upregulates GDF15 expression as an OS response.Through silencing of GDF15 and supplementation with recombinant GDF15 (rGDF15), we found that GDF15, expressed as an OS response, protected KGN cells against AA-induced OS effects, such as impairment of secretory and mitochondrial functions and apoptosis. Therefore, this study suggested that AA might induce OS in KGN cells and upregulate the expression of GDF15 as a response to OS.

Lipoxin (LTX A4 5S, 6R, 15R) levels drastically decrease after 5 years of hemodialysis treatment.

The increased risk of atherosclerosis in patients with chronic kidney disease (CKD) is associated with the increased concentration of fatty acids from the omega-6 family. Products of arachidonic acid oxidation, including prostaglandins, thromboxanes, hydroxyleicosa-tetraenoic acids (HETES) and hydroxyoctadecadienoic acids (HODES) are involved in the pathogenesis of cancer and cardiovascular diseases due to increased oxidative stress. The aim of our study was to determine the relations resulting from the duration of CKD treatment. One of our main concerns is, whether and when the cascade of synthesis of inflammatory mediators may be insufficient in patients with CKD during many years of treatment. The study involved 121 patients with CKD and 87 healthy volunteers. Eicosanoid profiles 9(S)-HODE, 13(S)-HODE, 5(S)-HETE, 12(S)-HETE, 15(S)-HETE, 5(S)-oxoETE, 16(RS)-HETE, and 5(S),6(R)-lipoxinA4, 5(S),6(R),15(R)-lipoxinA4 were extracted in plasma. The HPLC separations were performed by means of 1260 liquid chromatography. Patients with CKD have a significantly higher concentration of the following inflammatory mediators: 13(S)-HODE, 5(S)-HETE, 12(S)-HETE, 15(S)-HETE, 5(S)-oxoETE, 16(RS)-HETE, and 5(S),6(R), 15(R)-lipoxinA4 relative to the control group. However, the concentrations of 9(S)-HODE were lower in the CKD group. The comparison of sexes did not show significant differences in terms of CKD. A tendency for lower concentrations of HETE and HODE were observed in the group of men. 15LOX, 12LOX and 5LOX pathways in chronic kidney disease are increased, while COX are suppressed (9-HODE). The analysis of the treatment time of patients with CKD shows that incorrect levels of 5(S), 6(R) and 15(R)-lipoxinA4 are developed. We present a new evidence of possible concepts and future clinical interventions in patients suffering from chronic kidney disease for many years. These data for the first time demonstrate that lipoxin levels drastically decrease in the course of CKD. Therefore, synthetic LXA4 analogues may be used as an antioxidant therapy in CKD, which requires further research.

Analysis of eicosanoid oxidation products in Alzheimer brain by LC-MS with uniformly 13C-labeled internal standards.

The quantitative analysis of polyunsaturated fatty acyl (PUFA) chain oxidation products in tissue samples by mass spectrometry is hindered by the lack of durable internal standards for the large number of possible products. To address this problem in a study of oxidative PUFA degradation in Alzheimer's disease (AD) brain, uniformly 13C-labeled arachidonic acid (ARA) was produced biosynthetically, and allowed to oxidize under controlled conditions into a mixture of U-13C-labeled ARA oxidation products. The components of this mixture were characterized with respect to their partitioning behavior during lipid extraction, their durability during saponification, trends in mouse brain tissue concentrations during post mortem intervals, and their overall suitability as internal standards for multiple-reaction monitoring tandem mass spectrometry. This mixture has now been used as a set of internal standards to determine the relative abundance of ARA and 54 non-stereospecific oxidation products in milligram samples of brain tissue. Many of these oxidation products were recovered from both healthy mouse and healthy human brain, although some of them were unique to each source, and some have not heretofore been described. The list of oxidation products detected in AD brain tissue was the same as in healthy human brain, although simple hydroxy-eicosanoids were significantly increased in AD brain. while more complex oxidation products were not. These results are consistent with an increased level of chemically-mediated oxidative ARA degradation in Alzheimer's disease. However, they also point to the existence of processes that selectively produce or eliminate specific oxidation products, and those processes may account for some of the inconsistencies in previously reported results.

High levels of oxidized fatty acids in HDL are associated with impaired HDL function in patients with active rheumatoid arthritis.

The objective of this study was to evaluate oxidation products of arachidonic acid and linoleic acid in lipoproteins and synovial fluid (SF) from patients with active rheumatoid arthritis (RA) compared to non-RA controls. High-density lipoproteins (HDL) and low-density lipoproteins (LDL) were isolated from plasma using fast protein liquid chromatography and HDL was isolated from SF using dextran sulfate precipitation. 5-Hydroxyeicosatetraenoic acid (HETE), 12-HETE, 15-HETE, 9 hydroxyoctadecadienoic (HODE), and 13-HODE levels were measured in HDL, LDL, and SF by liquid chromatography-tandem mass spectrometry. HDL's anti-inflammatory function, cholesterol levels, myeloperoxidase (MPO) and paraoxonase 1 (PON1) activities were determined as previously. 5-HETE, 15-HETE, 9-HODE, and 13-HODE levels were significantly increased in HDL and LDL from patients with active RA (n=10) compared to healthy controls (n=8) and correlated significantly with measures of systemic inflammation, particularly in HDL (r=0.65-0.80, p values <0.004). Higher HETES and HODES in HDL were also significantly correlated with impaired HDL function as measured by the HDL inflammatory index (HII) (r=0.54-0.58; p values <0.03). 15-HETE levels and MPO activity were higher in RA SF (n=10) compared to osteoarthritis (OA) SF(n=11), and HDL from RA SF had worse function compared to OA SF HDL (HII=2.1±1.9 and 0.5±0.1), respectively (p<0.05). Oxidation products of arachidonic acid and linoleic acid are increased in HDL and LDL from patients with active RA compared to healthy controls, and are associated with worse anti-oxidant function of HDL. These results suggest a potential mechanism by which oxidative stress from active RA increases oxidized fatty acids in HDL, promoting HDL dysfunction, and thereby increasing atherosclerotic risk.

Absence of systemic oxidative stress and increased CSF prostaglandin F2α in progressive MS.

Objective:We aimed to investigate the role of oxidative stress in the progression of multiple sclerosis (MS).Methods:We determined by liquid chromatography–tandem mass spectrometry nonenzymatic (F2-isoprostanes) and enzymatic oxidation products of arachidonic acid (prostaglandin F2α [PGF2α]) in plasma and CSF of 45 controls (other neurologic disease [OND] with no signs of inflammation) and 62 patients with MS. Oxidation products were correlated with disease severity and validated biomarkers of inflammation (chemokine ligand 13; matrix metalloproteinase-9; osteopontin) and axonal damage (neurofilament light protein).Results:Compared with OND controls, plasma concentrations of F2-isoprostanes and PGF2α were significantly lower in patients with progressive disease, and decreased with increasing disability score (Expanded Disability Status Scale). In contrast, CSF concentrations of PGF2α, but not F2-isoprostanes, were significantly higher in patients with progressive disease than OND controls (p < 0.01). The content of PGF2α in CSF increased with disease severity (p = 0.044) and patient age (p = 0.022), although this increase could not be explained by age. CSF PGF2α decreased with natalizumab and methylprednisolone treatment and was unaffected by the use of nonsteroidal anti-inflammatory drug in secondary progressive MS. CSF PGF2α did not associate with validated CSF markers of inflammation and axonal damage that themselves did not associate with the Expanded Disability Status Scale.Conclusions:Our data suggest that MS progression is associated with low systemic oxidative activity. This may contribute to immune dysregulation with CNS inflammation accompanied by increased local cyclooxygenase-dependent lipid oxidation.

Bioactive Lipids Are Generated to Micromolar Levels during RBC Storage, Even in Leukoreduced Units

Background: The transfusion of stored red blood cells (RBCs) has been linked to negative outcomes, such as Transfusion-Related Acute Lung Injury (TRALI) and Transfusion Related Immunomodulation (TRIM). Bioactive lipids have been implicated as playing an important role in TRALI pathogenesis, both as a "second hit" when anti-HLA antibodies are present and also in anti-HLA independent TRALI. Likewise, bioactive lipids have been suggested to play a role in TRIM.Methods: To further study bioactive lipids in RBC storage, we developed a targeted metabolomics approach, with isotope dilution, to accurately and precisely quantify concentrations of multiple poly-unsaturated fatty acids (PUFAs) and their related oxidation products (oxylipins) in stored RBC units. Analytes in RBC samples were extracted using methanol with isotopically-labeled internal standards and analyzed by liquid chromatography-tandem mass spectrometry using multiple reaction monitoring (LC-MS/MS-MRM). Eight units of leukoreduced (LR) and non-leukoreduced (NLR) RBCs were analyzed longitudinally from 6 to 42 days of storage.Results: LR RBCs had a significant increase in multiple PUFAs over storage, including: arachidonic acid (AA), linoleic acid (LA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), linolenic acid (ALA), dihomo-γ-linolenic acid (DGLA), adrenic acid (22:4), and docosapentaenoic acid (22:5). Only a modest increase was observed over the first 20 days of storage, followed by a rapid increase from day 20 to day 42. AA showed the biggest increase in concentration, from 1.0±0.4 µM at day 6 to 13.4±5.2 µM at day 42 in LR RBC units. Oxidative products of PUFA metabolism were also analyzed. The concentration of hydroxyeicosatetraenoic acids (HETEs), oxidation products of AA, including 5-, 8-, 9-, 11-, 12- and 15-HETEs increased 5 to 13 fold from day 6 to day 42 of storage in LR RBCs. The concentration of other oxylipins including hydroxyoctadecadienoic acids(9- and 13-HODEs)and dihydroxyoctadecenoic acids (, 9, 10- and 12,13-DiHOMEs), hydroxyl-DHA(14-, 16- and 17-HDoHEs) were also significantly increased during storage. If present, prostaglandins (PGE2 and PGD2) and Leukotriene (LTB4), were below the lower limits of detection. Juxta position of LR and NLR RBCs, showed that the concentrations of 12-HETE, 15-HETE, 14-HDoHE, and 17-HDoHE in NLR units were significantly higher at day 42 (p<0.01). Of particular note, the average concentration of 12-HETE reached 1.1±0.8 µM in NLR at day 42, which was 45 fold higher than that in LR units. No differences in LR and NLR RBCs were noted at day 6.Conclusions: We report significant generation of PUFAs and products of lipid metabolism/oxidation over RBC storage, with a particular increase in generation after day 20 of storage. LR decreased the generation of some lipid species (12-HETE in particular); however, accumulation of many bioactive lipids occurs robustly despite LR. Because variation from unit to unit was small at day 6 but substantial at day 42, these findings also support significant donor-to-donor differences in the process of bioactive lipid generation during storage. Exactly how the generation of bioactive lipids effects medical outcomes of transfusion remains undetermined; however, the quantitation allowed by the current methods demonstrates the generation of bioactive lipids up to µM levels, which are known to be sufficient to have significant physiological and/or pathological impacts in other settings. DisclosuresZimring:Immucor Inc.: Research Funding; BloodworksNW: Patents & Royalties: Patent Application filed on technology in this abstract - no royalties.

Abstract 354: Vascular Oxidative Stress Promotes Aortic Stiffening In Hypertension

We have previously shown that T cells and the cytokine IL-17a contribute to aortic stiffening in hypertension. Because oxidative stress also contributes to hypertension, we sought to determine if chronic oxidative stress causes aortic stiffening and to determine if T cells are involved. We studied tgsm/p22phox mice, which have an overexpression of the NADPH oxidase subunit p22phox in the vascular smooth muscle and approximately 2-fold increase vascular reactive oxygen species (ROS). Six month-old tgsm/p22phox mice were normotensive at baseline but exhibited increased aortic stiffness as indicated by the leftward shift of stress-strain curves (p&lt;0.05). Aortic collagen content, as determined by hydroxyproline assay, was also increased in these mice compared to age-matched C57Bl/6 mice (76.3±11.9 vs. 43.2±11.9 ng/μg total protein, p&lt;0.01). At nine months of age, aortic stiffening did not further increase but tgsm/p22phox mice developed vascular inflammation and mild hypertension (137±9 systolic and 100±7 diastolic, p&lt;0.05), indicating that aortic stiffening precedes hypertension. Surprisingly, T cells isolated from the spleen of 9 month-old Tgsm/p22phox mice demonstrated marked production of IL-17a and IFN-gamma. To determine if inflammation contributes to these pathological events, we crossed the tgsm/p22phox mice with RAG-1-/- mice. The resultant mice were protected from vascular inflammation, aortic stiffening and hypertension. Tempol, a superoxide dismutase mimetic, normalized blood pressure (109±4 systolic and 95±4 diastolic, p&lt;0.05), reduced vascular collagen deposition (37.5±9.5 ng/μg total protein, p&lt;0.01) and prevented aortic stiffening in tgsm/p22phox mice. We have recently shown that isoketals, which are oxidation products of arachidonic acid, promote T cell activation. Treatment with the isoketal scavenger 2-hydroxybenzylamine also normalized blood pressure, prevented collagen deposition and aortic stiffening in 9 month-old tgsm/p22phox mice. These data are compatible with a pathway in which vascular oxidative stress leads to formation of isoketals, which promote T cell activation, production of cytokines such as IL-17a and IFN-gamma, which in turn promote collagen deposition and aortic stiffening.

Lipid Oxidation in Carriers of Lecithin:Cholesterol Acyltransferase Gene Mutations

Lecithin:cholesterol acyltransferase (LCAT) has been shown to play a role in the depletion of lipid oxidation products, but this has so far not been studied in humans. In this study, we investigated processes and parameters relevant to lipid oxidation in carriers of functional LCAT mutations. In 4 carriers of 2 mutant LCAT alleles, 63 heterozygotes, and 63 family controls, we measured activities of LCAT, paraoxonase 1, and platelet-activating factor-acetylhydrolase; levels of lysophosphatidylcholine molecular species, arachidonic and linoleic acids, and their oxidized derivatives; immunodetectable oxidized phospholipids on apolipoprotein (apo) B-containing and apo(a)-containing lipoproteins; IgM and IgG autoantibodies to malondialdehyde-low-density lipoprotein and IgG and IgM apoB-immune complexes; and the antioxidant capacity of high-density lipoprotein (HDL). In individuals with LCAT mutations, plasma LCAT activity, HDL cholesterol, apoA-I, arachidonic acid, and its oxidized derivatives, oxidized phospholipids on apo(a)-containing lipoproteins, HDL-associated platelet-activating factor-acetylhydrolase activity, and the antioxidative capacity of HDL were gene-dose-dependently decreased. Oxidized phospholipids on apoB-containing lipoproteins was increased in heterozygotes (17%; P<0.001) but not in carriers of 2 defective LCAT alleles. Carriers of LCAT mutations present with significant reductions in LCAT activity, HDL cholesterol, apoA-I, platelet-activating factor-acetylhydrolase activity, and antioxidative potential of HDL, but this is not associated with parameters of increased lipid peroxidation; we did not observe significant changes in the oxidation products of arachidonic acid and linoleic acid, immunoreactive oxidized phospholipids on apo(a)-containing lipoproteins, and IgM and IgG autoantibodies against malondialdehyde-low-density lipoprotein. These data indicate that plasma LCAT activity, HDL-associated platelet-activating factor-acetylhydrolase activity, and HDL cholesterol may not influence the levels of plasma lipid oxidation products.

Abstract 166: Activation of T Cells by Dendritic Cells in Hypertension: A Potential Role of Isoketal-modified Proteins

The adaptive immune system contributes to development of hypertension but the mechanisms or antigens involved are not known. Isoketals (IsoKs) are products of arachidonic acid oxidation that can cross-link lysine residues on proteins which in turn can be immunogenic. We sought to determine if IsoK-modified proteins serve as neoantigens presented by dendritic cells (DCs) to activate T cells in hypertension. Superoxide production by DCs was increased 5-fold by angiotensin II infusion compared to sham treated mice (334.0±49.7 versus 65.8±4.5 pmol/mg protein). This was NADPH-dependent as it did not occur in the gp91 phox-/- mice. This increase in superoxide was associated with accumulation of IsoKs in DCs and activation of DC IL-6 production (1411.0±1330.2 versus 576.2±512.8 pg/ml). Treatment with a potent IsoK scavenger, 2-hydroxybenzylamine, markedly attenuated angiotensin II-induced hypertension (131.4 ± 9.4 mmHg versus 160.1 ± 5.1 mmHg in control mice). Finally we employed an immunization assay involving priming by adoptive transfer of DCs from either sham or angiotensin II-treated mice to naïve recipients. T cells from these recipients were then cultured for 10 days with DCs from either sham or angiotensin II treated mice. DCs from angiotensin II treated mice, but not sham mice, caused a striking activation of CD8 + cells in this assay, as reflected by proliferation and polarization to Tc1 and Tc17 phenotypes. These studies show that angiotensin II-induced hypertension activates DCs, in large part by causing superoxide production and formation of IsoKs. We propose that IsoK-modified proteins can be presented as neoantigens by DCs, which in turn trigger T cell activation leading to hypertension.

Effect of Hypertension on Dendritic Cells and a potential role of Isoketals

We have previously shown that angiotensin II‐infusion activates dendritic cells (DCs) and that this contributes to blood pressure elevation. Reactive oxygen species (ROS) also contribute to hypertension, but the interaction of hypertensive stimuli, oxidative stress and DC activation are not defined. Isoketals are highly reactive oxidation products of arachidonic acid that cross‐link lysine residues on proteins. These can alter protein function and become antigenic. We sought to determine if hypertension alters DC ROS production and if isoketal formation is involved in hypertension. We found that angiotensin II infusion increased DC superoxide production by &gt;50%. Angiotensin II also caused a massive accumulation of isoketals in various tissues including the heart, kidney and vasculature. In additional experiments, we co‐administered salicylamine, a potent scavenger of isoketals. Salicylamine attenuated development of angiotensin II‐induced hypertension (142.59 ± 8.98 mmHg versus 175.53 ± 5.19 mmHg in control mice). These studies show that hypertension causes oxidative stress in DCs. Moreover, isoketal modified proteins may serve as neoantigens that trigger hypertension.

Elevated Plasma Oxidized Lipids in Severe Pulmonary Hypertension are Fully Restored by Estrogen Therapy

Recently we reported that estrogen (E2) rescues pre-existing severe pulmonary hypertension (PH). PH is associated with pulmonary vascular remodelling. Vascular disorders result from complex interactions between oxidized-lipoproteins, monocytes/macrophages, injured endothelium and smooth muscle cells. Biological oxidation products of arachidonic acid, including prostaglandins (PGs), thromboxanes (TXs), hydroxyeicosatetraenoic acids (HETEs) and hydroxyoctadecadienoic acids (HODEs) play an important role in the pathogenesis of atherosclerosis, yet their role in PH is not fully known. Here we investigated whether PH is associated with increased plasma levels of oxidized-lipids, and if E2 can restore their levels. To induce PH, rats received monocrotaline (MCT, 60mg/kg, s.c.). Severe PH was established 21-days after MCT (PH, n=8). E2 (42.5ug/kg/day, s.c., n=6) was given from day-21 to 30. Saline treated rats served as control (CTRL, n=7). Cardiac catheterization was performed terminally to record RV-pressure (RVP). Plasma HODEs, HETEs, TXs and PGs were determined by mass-spectrometry. p<0.05 was considered significant. Values were mean±SE. Rats developed severe PH [RVP=68±2 vs. 31±2 mmHg in CTRL, p<0.01). E2 rescued PH (RVP=38±1 mmHg, p<0.01 vs. PH). PH was associated with increased plasma levels (ng/ml of plasma) of 9-HODE (2217±381 vs. 949±255 in CTRL, p<0.05) and 13-HODE (4347±439 vs. 1871±609 in CTRL, p<0.05). E2 reversed PH-induced increase in HODEs (9-HODE=976±259; 13-HODE=2469±531; all p<0.05 vs. PH). PH led to significantly elevated plasma 5-HETE (938±77 vs. 663±15), 12-HETE (9720±972 vs. 6633±522) and 15-HETE (818±62 vs. 534±7; all p<0.05 vs. CTRL). E2 fully reversed the increase in HETEs (5-HETE=708±37; 12-HETE=6981±248; 15-HETE=622±50; p<0.05 vs. PH). PH also led to elevated plasma TXB2 (3.5±0.2 vs. 2.8±0.1), PGD2 (98±11 vs. 35±7) and PGE2 (27±1 vs. 13±2; all p<0.05 vs. CTRL) and E2 restored their levels (TXB2=2.6±0.2; PGD2=54±10; PGE2=19±2; all p<0.05). In conclusion, severe PH is associated with increased plasma levels of oxidized lipids that are fully restored by E2.

Regulation of the arachidonic acid mobilization in macrophages by combustion-derived particles

BackgroundAcute exposure to elevated levels of environmental particulate matter (PM) is associated with increasing morbidity and mortality rates. These adverse health effects, e.g. culminating in respiratory and cardiovascular diseases, have been demonstrated by a multitude of epidemiological studies. However, the underlying mechanisms relevant for toxicity are not completely understood. Especially the role of particle-induced reactive oxygen species (ROS), oxidative stress and inflammatory responses is of particular interest.In this in vitro study we examined the influence of particle-generated ROS on signalling pathways leading to activation of the arachidonic acid (AA) cascade. Incinerator fly ash particles (MAF02) were used as a model for real-life combustion-derived particulate matter. As macrophages, besides epithelial cells, are the major targets of particle actions in the lung murine RAW264.7 macrophages and primary human macrophages were investigated.ResultsThe interaction of fly ash particles with macrophages induced both the generation of ROS and as part of the cellular inflammatory responses a dose- and time-dependent increase of free AA, prostaglandin E2/thromboxane B2 (PGE2/TXB2), and 8-isoprostane, a non-enzymatically formed oxidation product of AA. Additionally, increased phosphorylation of the mitogen-activated protein kinases (MAPK) JNK1/2, p38 and ERK1/2 was observed, the latter of which was shown to be involved in MAF02-generated AA mobilization and phosphorylation of the cytosolic phospolipase A2. Using specific inhibitors for the different phospolipase A2 isoforms the MAF02-induced AA liberation was shown to be dependent on the cytosolic phospholipase A2, but not on the secretory and calcium-independent phospholipase A2. The initiation of the AA pathway due to MAF02 particle exposure was demonstrated to depend on the formation of ROS since the presence of the antioxidant N-acetyl-cysteine (NAC) prevented the MAF02-mediated enhancement of free AA, the subsequent conversion to PGE2/TXB2 via the induction of COX-2 and the ERK1/2 and JNK1/2 phosphorylation. Finally we showed that the particle-induced formation of ROS, liberation of AA and PGE2/TXB2 together with the phosphorylation of ERK1/2 and JNK1/2 proteins was decreased after pre-treatment of macrophages with the metal chelator deferoxamine mesylate (DFO).ConclusionsThese results indicate that one of the primary mechanism initiating inflammatory processes by incinerator fly ash particles seems to be the metal-mediated generation of ROS, which triggers via the MAPK cascade the activation of AA signalling pathway.

The effects of oxidation products of arachidonic acid and n3 fatty acids on vascular and platelet function

15-F2t-isoprostane (15-F2t-IsoP), an oxidation product of arachidonic acid (AA), affects vascular and platelet function; however, the bioactivity of other fatty acids oxidation products is unknown. This paper studied rat aortic vascular reactivity and human platelet aggregation in response to 14 oxidation products of AA, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and α-linolenic acid (ALA) compared with 15-F2t-IsoP. It also compared the F2t-IsoPs profile in human platelets. EPA-derived 15-F3t-IsoP constricted rat aorta less than 15-F2t-IsoP, but none of the other oxidation products affected vascular reactivity. Only 15-F2t-IsoP (10−4 M) directly affected platelet aggregation. 15-F3t-IsoP, ent-16-F1-phytoprostane (from ALA) and isofurans A and B (from AA) inhibited reversible aggregation to U46619. Unlike plasma, the platelet profile of F2-IsoP showed that 8-F2t-IsoP were higher than 15-F2t-IsoP. Unlike 15-F2t-IsoP, the test compounds derived from fatty acids oxidation did not affect vascular or platelet function. Elevated platelet 8-F2t-IsoP could limit 15-F2t-IsoP-induced aggregation under conditions of oxidant stress.

Isoprostanes

The isoprostanes (IsoPs) are a unique series of prostaglandin-like compounds formed in vivo via a nonenzymatic mechanism involving the free radical-initiated peroxidation of arachidonic acid. This article summarizes our current knowledge of these compounds. Herein, a historical account of their discovery and the mechanism of their formation are described. A specific class of IsoPs, the F2-IsoPs, are stable, robust molecules that can be measured as indices of endogenous oxidant stress. The utility of these molecules as biomarkers and methods by which these compounds can be quantified are discussed. In addition to the F2-IsoPs, isoprostanes with other prostane ring structures as well as oxidation products with furan and dioxolane rings can be generated from arachidonic acid. And, in more recent years, isoprostane-like compounds have been shown to be formed from polyunsaturated fatty acids including eicosapentaenoic acid [C20:5, omega-3], docosahexaenoic acid [C22:6, omega-3], and adrenic acid [C22:4, omega-6]. These findings will be summarized as well.

Pitfalls in the use of arachidonic acid oxidation products to assign lipoxygenase activity in cancer cells

Arachidonic acid (AA) reaction with cyclooxygenase (COX) and lipoxygenases (LOX) yield eicosanoids that can mediate prostate cancer proliferation and enhance both tumour vascularization and metastasis. Increasingly measurement of eicosanoids with liquid chromatography is employed to implicate LOX activity in different biological systems and in particular link LOX activity to the progression of cancer in experimental models. This study demonstrates that simply identifying patterns of eicosanoid regio-isomerism is insufficient to designate LOX activity in prostate cancer cells and the analysis must include complete stereochemical assignment of the various isomers in order to validate the assignment of LOX activity.

552: Elevated urinary isoprostanes in early pregnancy are associated with subsequent fetal growth restriction

Elevated amniotic fluid isoprostane levels have been demonstrated in pregnancies complicated by intrauterine growth restriction (IUGR). F2 isoprostanes are products of arachidonic acid oxidation resulting from free-radical attack of cell membrane phospholipids and circulating low density lipoproteins (LDLs). Urinary excretion of isoprostanes (8-iso-PGF2) is a reliable indicator of oxidative stress. We evaluated the role of oxidative stress in the pathophysiology of IUGR, using a non- invasive assay.

Crystal Structure of Inhibitor-Bound Human 5-Lipoxygenase-Activating Protein

Leukotrienes are proinflammatory products of arachidonic acid oxidation by 5-lipoxygenase that have been shown to be involved in respiratory and cardiovascular diseases. The integral membrane protein FLAP is essential for leukotriene biosynthesis. We describe the x-ray crystal structures of human FLAP in complex with two leukotriene biosynthesis inhibitors at 4.0 and 4.2 angstrom resolution, respectively. The structures show that inhibitors bind in membrane-embedded pockets of FLAP, which suggests how these inhibitors prevent arachidonic acid from binding to FLAP and subsequently being transferred to 5-lipoxygenase, thereby preventing leukotriene biosynthesis. This structural information provides a platform for the development of therapeutics for respiratory and cardiovascular diseases.