Reactive Gamma-ketoaldehydes Research Articles

Direct Detection of Isolevuglandins in Tissues using a D11 scFv-Alkaline Phosphatase Fusion Protein and Immunofluorescence.

Isolevuglandins (IsoLGs) are highly reactive gamma ketoaldehydes formed from H2-isoprostanes through lipid peroxidation and crosslink proteins leading to inflammation and various diseases including hypertension. Detection of IsoLG accumulation in tissues is crucial in shedding light on their involvement in the disease processes. However, measurement of IsoLGs in tissues is extremely difficult, and currently available tools, including mass spectrometry analysis, are laborious and extremely expensive. Here we describe a novel method for in situ detection of IsoLGs in tissues using alkaline phosphatase-conjugated D11 ScFv and a recombinant phage-display antibody produced in E. coli by immunofluorescent microscopy. Four controls were used for validating the staining: (1) staining with and without D11, (2) staining with bacterial periplasmic extract with the alkaline phosphatase linker, (3) irrelevant scFV antibody staining, and (4) competitive control with IsoLG prior to the staining. We demonstrate the effectiveness of the alkaline phosphatase-conjugated D11 in both human and mouse tissues with or without hypertension. This method will likely serve as an important tool to study the role of IsoLGs in a wide variety of disease processes.

Scavenging of highly reactive gamma-ketoaldehydes attenuates cognitive dysfunction associated with epileptogenesis

Cognitive dysfunction is a major comorbidity of the epilepsies; however, treatments targeting seizure-associated cognitive dysfunction, particularly deficits in learning and memory are not available. Isoketals and neuroketals, collectively known as gamma-ketoaldehydes are formed via the non-enzymatic, free radical catalyzed oxidation of arachidonic acid and docosahexaenoic acid, respectively. They are attractive candidates for oxidative protein damage and resultant cognitive dysfunction due to their formation within the plasma membrane and their high proclivity to form cytotoxic adducts on protein lysine residues. We tested the hypothesis that gamma-ketoaldehydes mechanistically contribute to seizure-associated memory impairment using a specific gamma-ketoaldehyde scavenger, salicylamine in the kainic acid and pilocarpine rat models of temporal lobe epilepsy. We show that gamma-ketoaldehydes are increased following epileptogenic injury in hippocampus and perirhinal cortex, two brain regions imperative for learning and memory. Treatment with an orally bioavailable, brain permeable scavenger, salicylamine attenuated 1) spatial memory deficits 2) reference memory deficits and 3) neuronal loss and astrogliosis in two mechanistically distinct models of epilepsy without affecting the epileptogenic injury or the development of chronic epilepsy. We have previously demonstrated that reactive oxygen species and the lipid peroxidation biomarkers, F2-isoprostanes are produced following status epilepticus. However, which reactive species specifically mediate oxidative damage to cellular macromolecules remains at large. We provide novel data suggesting that memory impairment occurs via gamma-ketoaldehyde production in two models of epilepsy and that treatment with a gamma-ketoaldehyde scavenger can protect vulnerable neurons. This work suggests a novel target and therapy to treat seizure-induced memory deficits in epilepsy.

Reactive gamma-ketoaldehydes as novel activators of hepatic stellate cells in vitro

AimsProducts of lipid oxidation, such as 4-hydroxynonenal (4-HNE), are key activators of hepatic stellate cells (HSC) to a pro-fibrogenic phenotype. Isolevuglandins (IsoLG) are a family of acyclic γ-ketoaldehydes formed through oxidation of arachidonic acid or as by-products of the cyclooxygenase pathway. IsoLGs are highly reactive aldehydes which are efficient at forming protein adducts and cross-links at concentrations 100-fold lower than 4-hydroxynonenal. Since the contribution of IsoLGs to liver injury has not been studied, we synthesized 15-E2-IsoLG and used it to investigate whether IsoLG could induce activation of HSC. ResultsPrimary human HSC were exposed to 15-E2-IsoLG for up to 48h. Exposure to 5μM 15-E2-IsoLG in HSCs promoted cytotoxicity and apoptosis. At non-cytotoxic doses (50 pM-500nM) 15-E2-IsoLG promoted HSC activation, indicated by increased expression of α-SMA, sustained activation of ERK and JNK signaling pathways, and increased mRNA and/or protein expression of cytokines and chemokines, which was blocked by inhibitors of JNK and NF-kB. In addition, IsoLG promoted formation of reactive oxygen species, and induced an early activation of ER stress, followed by autophagy. Inhibition of autophagy partially reduced the pro-inflammatory effects of IsoLG, suggesting that it might serve as a cytoprotective response. InnovationThis study is the first to describe the biological effects of IsoLG in primary HSC, the main drivers of hepatic fibrosis. ConclusionsIsoLGs represent a newly identified class of activators of HSC in vitro, which are biologically active at concentrations as low as 500 pM, and are particularly effective at promoting a pro-inflammatory response and autophagy.

P0437 : Reactive gamma-ketoaldehydes as novel activators of hepatic stellate cells in vitro

P0437 : Reactive gamma-ketoaldehydes as novel activators of hepatic stellate cells in vitro

Characterization of Scavengers of γ-Ketoaldehydes That Do Not Inhibit Prostaglandin Biosynthesis

Expression of cyclooxygenase-2 (COX-2) is associated with the development of many pathologic conditions. The product of COX-2, prostaglandin H(2) (PGH(2)), can spontaneously rearrange to form reactive gamma-ketoaldehydes called levuglandins (LGs). This gamma-ketoaldehyde structure confers a high degree of reactivity on the LGs, which rapidly form covalent adducts with primary amines of protein residues. Formation of LG adducts of proteins has been demonstrated in pathologic conditions (e.g., increased levels in the hippocampus in Alzheimer's disease) and during physiologic function (platelet activation). On the basis of knowledge that lipid modification of proteins is known to cause their translocation and to alter their function, we hypothesize that modification of proteins by LG could have functional consequences. Testing this hypothesis requires an experimental approach that discriminates between the effects of protein modification by LG and the effects of cyclooxygenase-derived prostanoids acting through their G-protein coupled receptors. To achieve this goal, we have synthesized and evaluated a series of scavengers that react with LG with a potency more than 2 orders of magnitude greater than that with the epsilon-amine of lysine. A subset of these scavengers are shown to block the formation of LG adducts of proteins in cells without inhibiting the catalytic activity of the cyclooxygenases. Ten of these selective scavengers did not produce cytotoxicity. These results demonstrate that small molecules can scavenge LGs in cells without interfering with the formation of prostaglandins. They also provide a working hypothesis for the development of pharmacologic agents that could be used in experimental animals in vivo to assess the pathophysiological contribution of levuglandins in diseases associated with cyclooxygenase up-regulation.

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.

Pyridoxamine Analogues Scavenge Lipid-Derived γ-Ketoaldehydes and Protect against H2O2-Mediated Cytotoxicity

Isoketals and levuglandins are highly reactive gamma-ketoaldehydes formed by oxygenation of arachidonic acid in settings of oxidative injury and cyclooxygenase activation, respectively. These compounds rapidly adduct to proteins via lysyl residues, which can alter protein structure/function. We examined whether pyridoxamine, which has been shown to scavenge alpha-ketoaldehydes formed by carbohydrate or lipid peroxidation, could also effectively protect proteins from the more reactive gamma-ketoaldehydes. Pyridoxamine prevented adduction of ovalbumin and also prevented inhibition of RNase A and glutathione reductase activity by the synthetic gamma-ketoaldehyde, 15-E2-isoketal. We identified the major products of the reaction of pyridoxamine with the 15-E2-isoketal, including a stable lactam adduct. Two lipophilic analogues of pyridoxamine, salicylamine and 5'-O-pentylpyridoxamine, also formed lactam adducts when reacted with 15-E2-isoketal. When we oxidized arachidonic acid in the presence of pyridoxamine or its analogues, pyridoxamine-isoketal adducts were found in significantly greater abundance than the pyridoxamine-N-acyl adducts formed by alpha-ketoaldehyde scavenging. Therefore, pyridoxamine and its analogues appear to preferentially scavenge gamma-ketoaldehydes. Both pyridoxamine and its lipophilic analogues inhibited the formation of lysyl-levuglandin adducts in platelets activated ex vivo with arachidonic acid. The two lipophilic pyridoxamine analogues provided significant protection against H2O2-mediated cytotoxicity in HepG2 cells. These results demonstrate the utility of pyridoxamine and lipophilic pyridoxamine analogues to assess the potential contributions of isoketals and levuglandins in oxidant injury and inflammation and suggest their potential utility as pharmaceutical agents in these conditions.

Prostaglandin H2‐derived adducts of proteins correlate with Alzheimer's disease severity

The formation of cyclooxygenase-derived lipid adducts of protein in brains of patients who had Alzheimer's disease has been investigated. The enzymatic product of the cyclooxygenases, prostaglandin H2, rearranges in part to highly reactive gamma-ketoaldehydes, levuglandin (LG) E(2) and LGD(2). These gamma-ketoaldehydes react with free amines on proteins to yield a covalent adduct. Utilizing analysis of the levuglandinyl-lysine adducts by liquid chromatography-tandem mass spectrometry, we now find that this post-translational modification is increased significantly in the hippocampus in Alzheimer's disease. The magnitude of the increase correlates with the pathological evidence of severity.

Isoprostanes: markers and mediators of oxidative stress

Some years ago it was discovered that prostaglandin F2-like compounds are formed in vivo by nonenzymatic free radical-catalyzed peroxidation of arachidonic acid. Because these compounds are a series of isomers that contain the prostane ring of prostaglandins, they were termed F2-isoprostanes. Intermediates in the isoprostane pathway are prostaglandin H2-like compounds that become reduced to form F2-isoprostanes but also undergo rearrangement in vivo to form E2-, D2-, A2-, J2-isoprostanes, isothromboxanes, and highly reactive gamma-ketoaldehydes, termed isoketals. Analogous compounds have also been shown to be formed from free radical mediated oxidation of docosoahexaenoic acid. Because docosahexaenoic acid is highly enriched in neurons, these compounds have been termed neuroprostanes and neuroketals. An important aspect of the discovery of isoprostanes is that measurement of F2-isoprostanes has emerged as one of the most reliable approaches to assess oxidative stress status in vivo, providing an important tool to explore the role of oxidative stress in the pathogenesis of human disease. Measurement of F4-neuroprostanes has also proved of value in exploring the role of oxidative stress in neurodegenerative diseases. Products of the isoprostane pathway have been found to exert potent biological actions and therefore may participate as physiological mediators of disease.

Covalent binding of isoketals to ethanolamine phospholipids

Free radicals have been strongly implicated in the pathogenesis of many human diseases. We previously identified the formation of highly reactive gamma-ketoaldehydes, isoketals, in vivo as products of free radical-induced peroxidation of arachidonic acid. Isoketals react with lysine residues on proteins at a rate that far exceeds that of 4-hydroxynonenal and demonstrate a unique proclivity to crosslink proteins. Hydroxynonenal has been shown to react with aminophospholipids, particularly phosphatidylethanolamine. We explored whether isoketals also react with phosphatidylethanolamine. Using liquid chromatography/electrospray mass spectrometry, we found that isoketals form pyrrole and Schiff base adducts with phosphatidylethanolamine. In addition, the ability of isoketals to covalently modify phosphatidylethanolamine is greater than that of 4-hydroxynonenal. These studies identify in vitro novel isoketal adducts. This provides the basis to explore the formation of isoketal-aminophospholipid adducts in vivo and the biological consequences of the formation of these adducts.

Isolevuglandins, a novel class of isoprostenoid derivatives, function as integrated sensors of oxidant stress and are generated by myeloperoxidase in vivo.

Isolevuglandins (isoLGs) are a family of reactive gamma-ketoaldehydes generated by free radical oxidation of arachidonate-containing lipids through the isoprostane pathway. Elevated plasma levels of isoLG protein adducts are observed in subjects with atherosclerosis compared with age/gender-matched controls. However, mechanisms for the generation of isoLGs in vivo are not established. Here we show that free radical-induced peroxidation promoted by the myeloperoxidase (MPO)/H2O2 system of leukocytes serves as one mechanism for the generation of isoLGs in vivo. Using a Candida sepsis model of inflammation, we demonstrate 3.5- and 2.7-fold increases in iso[4]LGE2 and isoLGE2 adducts of plasma proteins after pathogen exposure in wild-type mice. Plasma levels of F2 isoprostanes were not significantly increased after pathogen challenge in this model. MPO knockout mice demonstrated significant reductions (34%, P=0.003) in plasma levels of iso[4]LGE2 protein adducts after pathogen challenge compared with wild-type mice. Mass spectrometry and immunochemical methods demonstrate MPO-dependent formation of iso[4]LGE2 and isoLGE2 phospholipids and their corresponding isoLG protein adducts in model systems. The present studies thus identify MPO as one pathway for generation of isoLGs in vivo. They also suggest that long-lived protein isoLG adducts may serve as an alternative integrated sensor of oxidant stress in vivo.

Levuglandinyl Adducts of Proteins Are Formed via a Prostaglandin H2 Synthase-dependent Pathway after Platelet Activation

The product of oxygenation of arachidonic acid by the prostaglandin H synthases (PGHS), prostaglandin H(2) (PGH(2)), undergoes rearrangement to the highly reactive gamma-ketoaldehydes, levuglandin (LG) E(2), and LGD(2). We have demonstrated previously that LGE(2) reacts with the epsilon-amine of lysine to form both the levuglandinyl-lysine Schiff base and the pyrrole-derived levuglandinyl-lysine lactam adducts. We also have reported that these levuglandinyl-lysine adducts are formed on purified PGHSs following the oxygenation of arachidonic acid. We now present evidence that the levuglandinyl-lysine lactam adduct is formed in human platelets upon activation with exogenous arachidonic acid or thrombin. After proteolytic digestion of the platelet proteins, and isolation of the adducted amino acid residues, this adduct was identified by liquid chromatography-tandem mass spectrometry. We also demonstrate that formation of these adducts is inhibited by indomethacin, a PGHS inhibitor, and is enhanced by an inhibitor of thromboxane synthase. These data establish that levuglandinyl-lysine adducts are formed via a PGHS-dependent pathway in whole cells, even in the presence of an enzyme that metabolizes PGH(2). They also demonstrate that a physiological stimulus is sufficient to lead to the lipid modification of proteins through the levuglandin pathway in human platelets.

Identification of oxidized derivatives of neuroketals.

Oxidative stress and protein aggregation have been implicated in the pathogenesis of neurodegenerative diseases. The formation of neuroprostanes, isoprostane-like compounds formed from oxidation of docosahexaenoic acid, which is uniquely enriched in the brain, is increased in Alzheimer's disease. We recently identified the formation of a new class of highly reactive gamma-keto aldehydes, neuroketals, in vivo as products of the neuroprostane pathway. Neuroketals adduct to lysine residues of proteins with remarkable rapidity and induce cross-linking. Because neuroketals have either a 1,4-pentadiene or 1,4,7-octatriene side chain structure, we hypothesized that they could undergo further oxidation to form neuroketals with an additional hydroxyl group. Oxidation of docosahexaenoic acid in vitro yielded a series of compounds that were confirmed to be oxidized neuroketals by mass spectrometric analyses. Analysis of oxidized neuroketal adducts during oxidation of docosahexaenoic acid in the presence of lysine revealed the formation of oxidized Schiff base and hydroxylactam adducts. Oxidized hydroxylactam neuroketal-lysyl protein adducts, analyzed after digestion of proteins to individual amino acids, were not detected in nonoxidized rat brain synaptosomes but were readily detected following oxidation of synaptosomes. These studies indicate that neuroketals can undergo further oxidation, which in turn suggests that measurement of only unoxidized neuroketal adducts likely underestimates the amount of neuroketal adducts present in the brain in disorders of oxidant stress.

Prostaglandin H2 (PGH2) accelerates formation of amyloid beta1-42 oligomers.

Epidemiologic evidence implicates cyclooxygenase activity in the pathogenesis of Alzheimer's disease, in which amyloid plaques have been found to contain increased levels of dimers and higher multimers of the amyloid beta peptide. The product of the oxygenation of arachidonic acid by the cyclooxygenases, prostaglandin H2 (PGH2), rearranges non-enzymatically to several prostaglandins, including the highly reactive gamma-keto aldehydes, levuglandins E2 and D2. We demonstrate that PGH2 markedly accelerates the formation of dimers and higher oligomers of amyloid beta1-42. This is associated with the formation of levuglandin adducts of the peptide. These findings provide the molecular basis for a hypothesis linking cyclooxygenase activity to the formation of oligomers of amyloid beta.

Products of the isoprostane pathway: unique bioactive compounds and markers of lipid peroxidation.

We previously reported the discovery of prostaglandin F2-like compounds (F2-isoprostanes) formed by nonenzymatic free-radical-induced peroxidation of arachidonic acid. Quantification of F2-isoprostanes has proven to be a major advance in assessing oxidative stress status in vivo. Central in the pathway of formation of isoprostanes are prostaglandin H2-like endoperoxides, which also undergo rearrangement in vivo to form E-ring, D-ring, and thromboxane-ring compounds. E2- and D2-isoprostanes also undergo dehydration in vivo to form reactive cyclopentenone A2- and J2-isoprostanes, which are susceptible to Michael addition reactions with thiols. Recently, we described the formation of highly reactive gamma-ketoaldehydes (now termed isoketals) as products of isoprostane endoperoxide rearrangement which readily adduct to lysine residues on proteins and induce cross-links at rates that far exceed other aldehyde products of lipid peroxidation. Isoprostane-like compounds (neuroprostanes) and isoketal-like compounds (neuroketals) are formed from oxidation of docosahexaenoic acid, which is enriched in the brain, and measurement of neuroprostanes may provide a unique marker of oxidative neuronal injury.

Formation of Highly Reactive γ-Ketoaldehydes (Neuroketals) as Products of the Neuroprostane Pathway

Neuroprostanes are prostaglandin-like compounds produced by free radical-induced peroxidation of docosahexaenoic acid, which is highly enriched in the brain. We previously described the formation of highly reactive gamma-ketoaldehydes (isoketals) as products of the isoprostane pathway of free radical-induced peroxidation of arachidonic acid. We therefore explored whether isoketal-like compounds (neuroketals) are also formed via the neuroprostane pathway. Utilizing mass spectrometric analyses, neuroketals were found to be formed in abundance in vitro during oxidation of docosahexaenoic acid and were formed in greater abundance than isoketals during co-oxidation of docosahexaenoic and arachidonic acid. Neuroketals were shown to rapidly adduct to lysine, forming lactam and Schiff base adducts. Neuroketal lysyl-lactam protein adducts were detected in nonoxidized rat brain synaptosomes at a level of 0.09 ng/mg of protein, which increased 19-fold following oxidation in vitro. Neuroketal lysyl-lactam protein adducts were also detected in vivo in normal human brain at a level of 9.9 +/- 3.7 ng/g of brain tissue. These studies identify a new class of highly reactive molecules that may participate in the formation of protein adducts and protein-protein cross-links in neurodegenerative diseases and contribute to the injurious effects of other oxidative pathologies in the brain.

Characterization of the lysyl adducts of prostaglandin H-synthases that are derived from oxygenation of arachidonic acid.

These investigations characterize the covalent binding of reactive products of prostaglandin H-synthases (PGHSs) to the enzyme and to other molecules. The intermediate product of oxygenation of arachidonic acid by the PGHSs, prostaglandin (PG) H2, undergoes rearrangement to the highly reactive gamma-keto aldehydes, levuglandin (LG) E2 and D2. We previously have demonstrated that LGE2 reacts with the epsilon-amine of lysine to form both the lysyl-levuglandin Shiff base and the pyrrole-derived lysyl-levuglandin lactam adducts. We now demonstrate that these lysyl-levuglandin adducts are formed on the PGHSs following the oxygenation of arachidonic acid; after reduction of the putative Schiff base, proteolytic digestion of the enzyme, and isolation of the adducted amino acid residues, these adducts were identified by liquid chromatography-tandem mass spectrometry. The reactivity of the LGs is reflected by the finding that virtually all of the LG predicted to be formed from PGH2 can be accounted for as adducts of the PGH-synthase and that oxygenation of arachidonic acid by PGH-synthases also leads to the formation of adducts of other proteins present in the reaction solution. The reactivity of the PGH-synthase adducts themselves is demonstrated by the formation of intermolecular cross-links.

New developments in the isoprostane pathway: identification of novel highly reactive gamma-ketoaldehydes (isolevuglandins) and characterization of their protein adducts.

The bicyclic endoperoxide prostaglandin (PG) H2 undergoes nonenzymatic rearrangement not only to PGE2 and PGD2, but also to levuglandins (LG) E2 and D2, which are highly reactive gamma-ketoaldehydes. Isoprostanes (IsoPs) are PG-like compounds that are produced by nonenzymatic peroxidation of arachidonic acid. PGH2-like endoperoxides are intermediates in this pathway. Therefore, we explored whether the IsoP endoperoxides also undergo rearrangement to form IsoLGs. Oxidation of arachidonic acid in vitro resulted in the formation of abundant quantities of compounds that were established to be IsoLGs by using mass spectrometric analyses. However, the formation of IsoLGs could not be detected in biological systems subjected to an oxidant stress. We hypothesized that this was due to extremely rapid adduction of IsoLGs to proteins. This notion was supported by the finding that LGE2 adducted to albumin at a rate that exceeded that of 4-hydroxynonenal by several orders of magnitude: >50% of LGE2 had adducted within 20 s. We therefore undertook to characterize the nature of LG adducts. Using liquid chromatography electrospray tandem mass spectrometry, we established that LGs form oxidized pyrrole adducts (lactams and hydroxylactams) with the epsilon-amino group of lysine. Oxidation of low density lipoprotein resulted in readily detectable IsoLG adducts on apolipoprotein B after enzymatic digestion of the protein to individual amino acids. These studies identify a novel class of ketoaldehydes produced by the IsoP pathway that form covalent protein adducts at a rate that greatly exceeds that of other known aldehyde products of lipid peroxidation. Elucidation of the nature of the adducts formed by IsoLGs provides the basis to explore the formation of IsoLGs in vivo and investigate the potential biological ramifications of their formation in settings of oxidant injury.