Insight into the contribution of isoprostanoids to the health effects of omega 3 PUFAs.

Omega 3 polyunsaturated fatty acids have been reported to confer beneficial health effects notably in the field of cardiovascular and inflammatory diseases. The current knowledge suggests a significant portion of the effects of omega 3 polyunsaturated fatty acids are mediated by their oxygenated metabolites. This review attempts to cover the current literature about the contribution of specific omega 3 oxygenated metabolites, namely omega 3 isoprostanoids, which are produced through free-radical mediated oxidation. A special emphasis has been given to the most biologically relevant omega 3 polyunsaturated fatty acids namely the α-linolenic, eicosapentaenoic and docosahexaenoic acids. The review includes a comprehensive description of the biosynthetic pathways, a summary of studies related to the biological significance of omega 3 isoprostanoids as well as a critical description of analytical development in the field of omega 3 isoprostanoids profiling in biological samples.

The isoprostanes--25 years later.

Isoprostanes (IsoPs) are prostaglandin-like molecules generated independent of the cyclooxygenase (COX) by the free radical-induced peroxidation of arachidonic acid. The first isoprostane species discovered were isomeric to prostaglandin F2α and were thus termed F2-IsoPs. Since the initial discovery of the F2-IsoPs, IsoPs with differing ring structures have been identified as well as IsoPs from different polyunsaturated fatty acids, including eicosapentaenoic acid and docosahexanenoic acid. The discovery of these molecules in vivo in humans has been a major contribution to the field of lipid oxidation and free radical research over the course of the past 25 years. These molecules have been determined to be both biomarkers and mediators of oxidative stress in numerous disease settings. This review focuses on recent developments in the field with an emphasis on clinical research. Special focus is given to the use of IsoPs as biomarkers in obesity, ischemia-reperfusion injury, the central nervous system, cancer, and genetic disorders. Additionally, attention is paid to diet and lifestyle factors that can affect endogenous levels of IsoPs. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance."

NADPH:quinone oxidoreductase 1 regulates host susceptibility to ozone via isoprostane generation.

NADPH:quinone oxidoreductase 1 (NQO1) is recognized as a major susceptibility gene for ozone-induced pulmonary toxicity. In the absence of NQO1 as can occur by genetic mutation, the human airway is protected from harmful effects of ozone. We recently reported that NQO1-null mice are protected from airway hyperresponsiveness and pulmonary inflammation following ozone exposure. However, NQO1 regenerates intracellular antioxidants and therefore should protect the individual from oxidative stress. To explain this paradox, we tested whether in the absence of NQO1 ozone exposure results in increased generation of A(2)-isoprostane, a cyclopentenone isoprostane that blunts inflammation. Using GC-MS, we found that NQO1-null mice had greater lung tissue levels of D(2)- and E(2)-isoprostanes, the precursors of J(2)- and A(2)-isoprostanes, both at base line and following ozone exposure compared with congenic wild-type mice. We confirmed in primary cultures of normal human bronchial epithelial cells that A(2)-isoprostane inhibited ozone-induced NF-κB activation and IL-8 regulation. Furthermore, we determined that A(2)-isoprostane covalently modified the active Cys(179) domain in inhibitory κB kinase in the presence of ozone in vitro, thus establishing the biochemical basis for A(2)-isoprostane inhibition of NF-κB. Our results demonstrate that host factors may regulate pulmonary susceptibility to ozone by regulating the generation of A(2)-isoprostanes in the lung. These observations provide the biochemical basis for the epidemiologic observation that NQO1 regulates pulmonary susceptibility to ozone.

Discovery of a highly potent anti-inflammatory epoxyisoprostane-derived lactone.

Epoxyisoprostanes EI (1) and EC (2) are effective inhibitors of the secretion of proinflammatory cytokines IL-6 and IL-12. In detailed studies toward the investigation of the molecular mode of action of these structures, a highly potent lactone (3) derived from 1 was identified. The known isoprostanoids 1 and 2 are most likely precursors of 3, the product of facile intramolecular reaction between the epoxide with the carboxylic acid in 2.

Evidence for the importance of OxPAPC interaction with cysteines in regulating endothelial cell function.

Oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycerol-3-phosphatidylcholine (PAPC), referred to as OxPAPC, and an active component, 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphatidylcholine (PEIPC), accumulate in atherosclerotic lesions and regulate over 1,000 genes in human aortic endothelial cells (HAEC). We previously demonstrated that OxPNB, a biotinylated analog of OxPAPC, covalently binds to a number of proteins in HAEC. The goal of these studies was to gain insight into the binding mechanism and determine whether binding regulates activity. In whole cells, N-acetylcysteine inhibited gene regulation by OxPAPC, and blocking cell cysteines with N-ethylmaleimide strongly inhibited the binding of OxPNB to HAEC proteins. Using MS, we demonstrate that most of the binding of OxPAPC to cysteine is mediated by PEIPC. We also show that OxPNB and PEIPE-NB, the analog of PEIPC, bound to a model protein, H-Ras, at cysteines previously shown to regulate activity in response to 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2). This binding was observed with recombinant protein and in cells overexpressing H-Ras. OxPAPC and PEIPC compete with OxPNB for binding to H-Ras. 15dPGJ2 and OxPAPC increased H-Ras activity at comparable concentrations. Using microarray analysis, we demonstrate a considerable overlap of gene regulation by OxPAPC, PEIPC, and 15dPGJ2 in HAEC, suggesting that some effects attributed to 15dPGJ2 may also be regulated by PEIPC because both molecules accumulate in inflammatory sites. Overall, we provide evidence for the importance of OxPAPC-cysteine interactions in regulating HAEC function.

The neurobiology of isoprostanes and Alzheimer's disease.

In its sporadic form Alzheimer's disease (AD) results from a combination of genetic and environmental risk factors with abnormal oxidative reactions, which result in free radical mediated injury of the brain. Isoprostanes are oxidized lipids formed by a free radical mediated mechanism, which in recent years have emerged as a reliable and sensitive marker of lipid peroxidation and oxidative stress. Consistent data show that they are increased in the brain of human AD as well as AD animal models. Besides their role as biomarkers, isoprostanes possess important biological effects, functioning as mediators of the cellular response to oxidative stress within the CNS. Recent evidence indicates that these lipid oxidation products, by activating the thromboxane receptor system, mediate the pro-amyloidotic neuronal response to oxidative stress in an experimental model of AD. This novel observation has important clinical implication, since pharmacologic modulation of the TP receptor system by selective antagonists, some of which are already available, could represent a novel therapeutic opportunity for AD as disease-modifying agents.

The fatty acid oxidation product 15-A3t-isoprostane is a potent inhibitor of NFκB transcription and macrophage transformation.

Fatty acids such as eicosapentaenoic acid (EPA) have been shown to be beneficial for neurological function and human health. It is widely thought that oxidation products of EPA are responsible for biological activity, although the specific EPA peroxidation product(s) which exert these responses have not yet been identified. In this work we provide the first evidence that the synthesized representative cyclopentenone IsoP, 15-A(3t)-IsoP, serves as a potent inhibitor of lipopolysaccharide-stimulated macrophage activation. The anti-inflammatory activities of 15-A(3t)-IsoP were observed in response not only to lipopolysaccharide, but also to tumor necrosis factor alpha and IL-1b stimulation. Subsequently, this response blocked the ability of these compounds to stimulate nuclear factor kappa b (NFκB) activation and production of proinflammatory cytokines. The bioactivity of 15-A(3t)-IsoP was shown to be dependent upon an unsaturated carbonyl residue which transiently adducts to free thiols. Site directed mutagenesis of the redox sensitive C179 site of the Ikappa kinase beta subunit, blocked the biological activity of 15-A(3t)-IsoP and NFκB activation. The vasoprotective potential of 15-A(3t)-IsoP was underscored by the ability of this compound to block oxidized lipid accumulation, a critical step in foam cell transformation and atherosclerotic plaque formation. Taken together, these are the first data identifying the biological activity of a specific product of EPA peroxidation, which is formed in abundance in vivo. The clear mechanism linking 15-A(3t)-IsoP to redox control of NFκB transcription, and the compound's ability to block foam cell transformation suggest that 15-A(3t)-IsoP provides a unique and potent tool to provide vaso- and cytoprotection under conditions of oxidative stress.

Quantification of 15-F2t-isoprostane in human plasma and urine: results from enzyme-linked immunoassay and liquid chromatography/tandem mass spectrometry cannot be compared.

Quantification of F(2)-isoprostanes is considered a reliable index of the oxidative stress status in vivo. Several immunoassays and chromatography/mass spectrometry-based assays are available for 15-F(2t)-isoprostane quantification. However, it remains unclear if results of immunoassays using different assays can be compared with those of liquid chromatography/mass spectrometry (LC/MS) assays. Previous studies comparing enzyme-linked immunosorbent assay (ELISA) and more specific gas chromatography/mass spectrometry assays have already indicated that ELISAs may overestimate 15-F(2t)-isoprostane concentrations in human plasma. Concentrations of 15-F(2t)-isoprostane in 25 human plasma and urine samples were measured by three commercially available ELISA assays (Assay Designs, Cayman Chemical and Oxford Biomedical Research) and compared with the concentrations measured with a validated, semi-automated high-throughput HPLC tandem mass spectrometry assay (LC/LC-MS/MS). All three ELISAs measured substantially higher 15-F(2t)-isoprostane concentrations (2.1-182.2-fold higher in plasma; 0.4-61.9-fold higher in urine) than LC/LC-MS/MS. Utilization of solid-phase extraction (SPE) columns, especially isoprostane affinity purification columns, brought ELISA isoprostane urine concentrations closer to the LC/LC-MS/MS results. However, SPE did not have much of an effect on ELISA plasma concentrations which remained significantly higher than corresponding LC/LC-MS/MS results. A poor correlation not only between LC/LC-MS/MS and immunoassay results, but also among the immunoassays was found. Especially in plasma, ELISAs grossly overestimate 15-F(2t)-isoprostane concentrations and are not comparable with each other or with LC/LC-MS/MS. It is most disturbing that a sample with relatively high concentrations measured with one ELISA may show low concentrations with another ELISA, and vice versa, potentially affecting the conclusions drawn from such data. The use of specific mass spectrometry-based assays seems advisable.

Platelet function and Isoprostane biology. Should isoprostanes be the newest member of the orphan-ligand family?

While there have been many reports investigating the biological activity and signaling mechanisms of isoprostanes, their role in biology, particularly in platelets, appears to still be underestimated. Moreover, whether these lipids have their own receptors is still debated, despite multiple reports that discrete receptors for isoprostane do exist on platelets, vascular tissues, amongst others. This paper provides a review of the important literature of isoprostanes and provides reasoning that isoprostanes should be classified as orphan ligands until their receptor(s) is/are identified.

Lipase-catalyzed regioselective monoacetylation of unsymmetrical 1,5-primary diols.

Lipase B from Candida antarctica (CALB) has been selected as the most suitable enzyme to catalyze the regioselective monoacetylation of 1,5-diol isoprostane intermediate, using vinyl acetate as an acyl transfer reagent in THF. We next applied this reaction on linear 2-substituted, 2,2'-disubstituted-1,5-pentanediols, and cyclic 2,3-disubstituted-1,5-pentanediols. To rationalize the regioselectivity observed, molecular docking simulations were performed.

Total synthesis of 15-D(2t)- and 15-epi-15-E(2t)-isoprostanes.

The first total synthesis of 15-D(2t)-isoprostane is described. (-)-(9S,15S)-15-D(2t)-IsoP 1 and (+)-(11R,15R)-15-epi-15-E(2t)-IsoP 2 have been obtained in 15 steps from orthogonally protected enantiopure bicycle 3. Key features include an easy introduction of the cis side chains via ozonolysis, a highly selective enzymatic chemical differentiation of a non-meso-1,5-diol, and the use of a common synthetic intermediate allowing a stereodivergent approach to the target molecules.

Toward the elucidation of the metabolism of 15-E(2)-isoprostane: the total synthesis of the methyl ester of a potential central metabolite.

An 11-step total synthesis of the methyl ester of a potential metabolite of the autoxidatively formed natural product 15-E(2)-IsoP, whose metabolism is not known, is reported. Several vinylogous Mukaiyama aldol additions were tested for the assembly of the acyclic C7-C20 precursor. A new oxidative dianion cyclization served to access the cyclopentane core. The full carbon skeleton was synthesized by an acetylide alkylation. The overall yield of the metabolite amounts to 1.4% for the most efficient route. The results demonstrate convincingly that E(2)-IsoP metabolites are highly epimerization-sensitive and that they may thus also contribute to PGE(2)-action and metabolism.

Opposite effects of anandamide and N-arachidonoyl dopamine in the regulation of prostaglandin E and 8-iso-PGF formation in primary glial cells.

It is widely accepted that neuroinflammation is a key player in various pathological events associated with brain injury. More specifically, glial activation and the subsequent release of pro-inflammatory cytokines, reactive oxygen species (ROS), and prostaglandins play a role of paramount importance in cerebral damage. In this study, we examined the role of two endocannabinoids, anandamide (AEA) and N-arachidonoyldopamine (NADA) in the regulation of prostaglandin E(2) (PGE(2)) synthesis in primary glial cells. We show that NADA is a potent inhibitor of PGE(2) synthesis in lipopolysaccharide (LPS) stimulated cells, without modifying the expression or enzymatic activity of COX-2 and the production of prostaglandin D(2). We also show that NADA has the ability to prevent the free radical formation in primary microglial cells. The key findings of this investigation are our observation that AEA and NADA have opposite effects on glial cells and, most importantly, the first description of NADA as a potential antioxidative and anti-inflammatory agent acting through a mechanism that involves reduction in the synthesis of microsomal prostaglandin E synthase in LPS-activated microglia. These findings provide new mechanistic insights into the anti-inflammatory activities of NADA in the CNS and its potential to design novel therapeutic strategies to manage neuroinflammatory diseases.

Biological effect of protein modifications by lipid peroxidation products.

The products of lipid peroxidation, resulting from cell metabolism as well as the action of external physical factors and xenobiotics, have a significant impact on cell functions. One of the mechanisms by which lipid peroxidation products influence cells is the formation of adducts with proteins, including enzymes and signaling molecules. This review describes the biological consequences of protein adduct formation with oxidative lipid fragmentation products such as 4-hydroxynonenal (4-HNE), malondialdehyde (MDA), and acrolein, as well as cyclization products including isoprostanes, isoketals, and isolevuglandins. The generation of protein adducts with lipid peroxidation products can stimulate the antioxidant system, which may also possess proinflammatory or proapoptotic effects. However, the role of adducts between lipid peroxidation products and proteins depends on the condition of the cells and can range from the function of cytoprotective activity stimulation, to induction of toxicity involved in the development of degenerative diseases.

Assessment of lipid peroxidation and artificial neural network models in early Alzheimer Disease diagnosis.

OBJECTIVE: Lipid peroxidation constitutes a molecular mechanism involved in early Alzheimer Disease (AD) stages, and artificial neural network (ANN) analysis is a promising non-linear regression model, characterized by its high flexibility and utility in clinical diagnosis. ANN simulates neuron learning procedures and it could provide good diagnostic performances in this complex and heterogeneous disease compared with linear regression analysis. DESIGN AND METHODS: In our study, a new set of lipid peroxidation compounds were determined in urine and plasma samples from patients diagnosed with early Alzheimer Disease (n = 70) and healthy controls (n = 26) by means of ultra-performance liquid chromatography coupled with tandem mass-spectrometry. Then, a model based on ANN was developed to classify groups of participants. RESULTS: The diagnostic performances obtained using an ANN model for each biological matrix were compared with the corresponding linear regression model based on partial least squares (PLS), and with the non-linear (radial and polynomial) support vector machine (SVM) models. Better accuracy, in terms of receiver operating characteristic-area under curve (ROC-AUC), was obtained for the ANN models (ROC-AUC 0.882 in plasma and 0.839 in urine) than for PLS and SVM models. CONCLUSION: Lipid peroxidation and ANN constitute a useful approach to establish a reliable diagnosis when the prognosis is complex, multidimensional and non-linear.

Lipidomic methodologies for biomarkers of chronic inflammation in nutritional research: ω-3 and ω-6 lipid mediators.

The evolutionary history of hominins has been characterized by significant dietary changes, which include the introduction of meat eating, cooking, and the changes associated with plant and animal domestication. The Western pattern diet has been linked with the onset of chronic inflammation, and serious health problems including obesity, metabolic syndrome, and cardiovascular diseases. Diets enriched with ω-3 marine PUFAs have revealed additional improvements in health status associated to a reduction of proinflammatory ω-3 and ω-6 lipid mediators. Lipid mediators are produced from enzymatic and non-enzymatic oxidation of PUFAs. Interest in better understanding the occurrence of these metabolites has increased exponentially as a result of the growing evidence of their role on inflammatory processes, control of the immune system, cell signaling, onset of metabolic diseases, or even cancer. The scope of this review has been to highlight the recent findings on: a) the formation of lipid mediators and their role in different inflammatory and metabolic conditions, b) the direct use of lipid mediators as antiinflammatory drugs or the potential of new drugs as a new therapeutic option for the synthesis of antiinflammatory or resolving lipid mediators and c) the impact of nutritional interventions to modulate lipid mediators synthesis towards antiinflammatory conditions. In a second part, we have summarized methodological approaches (Lipidomics) for the accurate analysis of lipid mediators. Although several techniques have been used, most authors preferred the combination of SPE with LC-MS. Advantages and disadvantages of each method are herein addressed, as well as the main LC-MS difficulties and challenges for the establishment of new biomarkers and standardization of experimental designs, and finally to deepen the study of mechanisms involved on the inflammatory response.

E-Ring Isoprostanes Stimulate a Cl Conductance in Airway Epithelium via Prostaglandin E2-Selective Prostanoid Receptors.

Isoprostanes comprise a class of membrane lipid metabolites produced during oxidative stress, including asthma, chronic obstructive pulmonary disease, and cystic fibrosis. They are widely recognized to evoke a variety of biological responses in airway and pulmonary vascular smooth muscle, lymphatics, and innervation. However, their effects on airway epithelium are largely unstudied. We examined the electrophysiological responses evoked by several different isoprostane species in bovine airway epithelium using the Ussing chamber technique. The E-ring isoprostanes 15-E(1t)-IsoP and 15-E(2t)-IsoP evoked a substantial increase in short-circuit current (I(SC)), whereas four different F-ring isomers were ineffective. 15-E(2t)-IsoP-evoked I(SC) was mimicked by the prostaglandin E(2)-selective prostanoid receptor (EP)-agonist prostaglandin E(2) but not by agonists of EP(1)/EP(3)-, FP-, or TP receptors (sulprostone, fluprostenol, and U46619, respectively). This response was significantly reduced by the EP(4)-receptor blocker GW627386 but not by blockers of other prostanoid receptors (ICI 192,605 [TP-selective], SC19220 [EP(1)-selective], AH6809 [DP/EP(1)/EP(2)-selective], and AL8810 [FP-selective]). 15-E(2t)-IsoP-evoked I(SC) was reduced by blockers of Cl(-) channels (niflumic acid and 5-nitro-2-(3-phenylpropylamino)-benzoic acid), of Na(+)/K(+)/2Cl(-) co-transport (furosemide and bumetanide), of adenylate cyclase (MDL 12,330A), or of guanylate cyclase (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) but not by blockers of Na(+) conductances (amiloride). We conclude that 15-E(2t)-IsoP activates a transepithelial Cl(-) conductance in bovine airway epithelium through an EP(4) receptor coupled to adenylate cyclase and soluble guanylate cyclase.

Eicosapentaenoic-acid-derived isoprostanes: synthesis and discovery of two major isoprostanes.

The stereospecific synthesis of two all-syn-EPA-derived isoprostanes (iPs), 5-epi-8,12-iso-iPF(3alpha)-VI 17 and 8,12-iso-iPF(3alpha)-VI 18, has been accomplished. These two synthetic probes have been used to discover and identify their presence in human urine. The eventual quantitative measurement of these two iPs may be a valuable index of oxidative stress in people with eicosapentaenoic acid- (EPA) and docosahexaenoic acid- (DHA) enriched phospholipids.

Isoprostanoids quantitative profiling of marine red and brown macroalgae.

With the increasing demand for direct human and animal consumption seaweed farming is rapidly expanding worldwide. Macroalgae have colonized aquatic environments in which they are submitted to frequent changes in biotic and abiotic factors that can trigger oxidative stress (OS). Considering that isoprostanoid derivatives may constitute the most relevant OS biomarkers, we were interested to establish their profile in two red and four brown macroalgae. Seven phytoprostanes, three phytofuranes, and four isoprostanes were quantified through a new micro-LC-MS/MS method. The isoprostanoid contents vary greatly among all the samples, the ent-16(RS)-9-epi-ST-Δ14-10-PhytoF and the sum of 5-F2t-IsoP and 5-epi-5F2t-IsoP being the major compounds for most of the macroalgae studied. We further quantified these isoprostanoids in macroalgae submitted to heavy metal (copper) exposure. In most of the cases, their concentrations increased after 24 h of copper stress corroborating the original hypothesis. One exception is the decrease of ent-9-L1-PhytoP content in L. digitata.