Plasma oxylipin profiles reflect Parkinson's disease stage.

Derivatives of polyunsaturated fatty acids (PUFAs), also known as oxylipins, are key participants in regulating inflammation. Neuroinflammation is involved in many neurodegenerative diseases, including Parkinson's disease. The development of ultra-high-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) facilitated the study of oxylipins on a system level, i.e., the analysis of oxylipin profiles. We analyzed oxylipin profiles in the blood plasma of 36 healthy volunteers (HC) and 73 patients with Parkinson's disease (PD), divided into early (L\M, 29 patients) or advanced (H, 44 patients) stages based on the Hoehn and Yahr scale. Among the 40 oxylipins detected, we observed a decrease in the concentration of arachidonic acid (AA) and AA derivatives, including anandamide (AEA) and Leukotriene E4 (LTE4), and an increase in the concentration of hydroxyeicosatetraenoic acids 19-HETE and 12-HETE (PD vs HC). Correlation analysis of gender, age of PD onset, and disease stages revealed 20 compounds the concentration of which changed depending on disease stage. Comparison of the acquired oxylipin profiles to openly available PD patient brain transcriptome datasets showed that plasma oxylipins do not appear to directly reflect changes in brain metabolism at different disease stages. However, both the L\M and H stages are characterized by their own oxylipin profiles - in patients with the H stage oxylipin synthesis is increased, while in patients with L\M stages oxylipin synthesis decreases compared to HC. This suggests that different therapeutic approaches may be more effective for patients at early versus late stages of PD.

Targeted Lipidomic Analysis of Aqueous Humor Reveals Signaling Lipid-Mediated Pathways in Primary Open-Angle Glaucoma.

Primary open-angle glaucoma (POAG) is characterized by degeneration of retinal ganglion cells associated with an increase in intraocular pressure (IOP) due to hindered aqueous humor (AH) drainage through the trabecular meshwork and uveoscleral pathway. Polyunsaturated fatty acids and oxylipins are signaling lipids regulating neuroinflammation, neuronal survival and AH outflow. Among them, prostaglandins have been previously implicated in glaucoma and employed for its treatment. This study addressed the role of signaling lipids in glaucoma by determining their changes in AH accompanying IOP growth and progression of the disease. Eye liquids were collected from patients with POAG of different stages and cataract patients without glaucoma. Lipids were identified and quantified by UPLC-MS/MS. The compounds discriminating glaucoma groups were recognized using ANCOVA and PLS-DA statistic approaches and their biosynthetic pathways were predicted by bioinformatics. Among 22 signaling lipids identified in AH, stage/IOP-dependent alterations in glaucoma were provided by a small set of mediators, including 12,13-DiHOME, 9- and 13-HODE/KODE, arachidonic acid and lyso-PAF. These observations correlated with the expression of cytochromes P450 (CYPs) and phospholipases A2 in the ocular tissues. Interestingly, tear fluid exhibited similar lipidomic alterations in POAG. Overall, POAG may involve arachidonic acid/PAF-dependent pathways and oxidative stress as evidenced from an increase in its markers, KODEs and 12,13-DiHOME. The latter is a product of CYPs, one of which, CYP1B1, is known as POAG and primary congenital glaucoma-associated gene. These data provide novel targets for glaucoma treatment. Oxylipin content of tear fluid may have diagnostic value in POAG.

Inflammation in Dry Eye Syndrome: Identification and Targeting of Oxylipin-Mediated Mechanisms.

Dry eye syndrome (DES) is characterized by decreased tear production and stability, leading to desiccating stress, inflammation and corneal damage. DES treatment may involve targeting the contributing inflammatory pathways mediated by polyunsaturated fatty acids and their derivatives, oxylipins. Here, using an animal model of general anesthesia-induced DES, we addressed these pathways by characterizing inflammatory changes in tear lipidome, in correlation with pathophysiological and biochemical signs of the disease. The decline in tear production was associated with the infiltration of inflammatory cells in the corneal stroma, which manifested one to three days after anesthesia, accompanied by changes in tear antioxidants and cytokines, resulting in persistent damage to the corneal epithelium. The inflammatory response manifested in the tear fluid as a short-term increase in linoleic and alpha-linolenic acid-derived oxylipins, followed by elevation in arachidonic acid and its derivatives, leukotriene B4 (5-lipoxigenase product), 12-hydroxyeicosatetraenoic acid (12-lipoxigeanse product) and prostaglandins, D2, E2 and F2α (cyclooxygenase products) that was observed for up to 7 days. Given these data, DES was treated by a novel ophthalmic formulation containing a dimethyl sulfoxide-based solution of zileuton, an inhibitor of 5-lipoxigenase and arachidonic acid release. The therapy markedly improved the corneal state in DES by attenuating cytokine- and oxylipin-mediated inflammatory responses, without affecting tear production rates. Interestingly, the high efficacy of the proposed therapy resulted from the synergetic action of its components, namely, the general healing activity of dimethyl sulfoxide, suppressing prostaglandins and the more specific effect of zileuton, downregulating leukotriene B4 (inhibition of T-cell recruitment), as well as upregulating docosahexaenoic acid (activation of resolution pathways).

Oxylipin Profiles in Plasma of Patients with Wilson's Disease.

Wilson's disease (WD) is a rare autosomal recessive metabolic disorder resulting from mutations in the copper-transporting, P-type ATPase gene ATP7B gene, but influences of epigenetics, environment, age, and sex-related factors on the WD phenotype complicate diagnosis and clinical manifestations. Oxylipins, derivatives of omega-3, and omega-6 polyunsaturated fatty acids (PUFAs) are signaling mediators that are deeply involved in innate immunity responses; the regulation of inflammatory responses, including acute and chronic inflammation; and other disturbances related to any system diseases. Therefore, oxylipin profile tests are attractive for the diagnosis of WD. With UPLC-MS/MS lipidomics analysis, we detected 43 oxylipins in the plasma profiles of 39 patients with various clinical manifestations of WD compared with 16 healthy controls (HCs). Analyzing the similarity matrix of oxylipin profiles allowed us to cluster patients into three groups. Analysis of the data by VolcanoPlot and partial least square discriminant analysis (PLS-DA) showed that eight oxylipins and lipids stand for the variance between WD and HCs: eicosapentaenoic acid EPA, oleoylethanolamide OEA, octadecadienoic acids 9-HODE, 9-KODE, 12-hydroxyheptadecatrenoic acid 12-HHT, prostaglandins PGD2, PGE2, and 14,15-dihydroxyeicosatrienoic acids 14,15-DHET. The compounds indicate the involvement of oxidative stress damage, inflammatory processes, and peroxisome proliferator-activated receptor (PPAR) signaling pathways in this disease. The data reveal novel possible therapeutic targets and intervention strategies for treating WD.

Comparative lipidomic analysis of inflammatory mediators in the aqueous humor and tear fluid of humans and rabbits.

INTRODUCTION: Ocular inflammation is a key pathogenic factor in most blindness-causing visual disorders. It can manifest in the aqueous humor (AH) and tear fluid (TF) as alterations in polyunsaturated fatty acids (PUFAs) and their metabolites, oxylipins, lipid mediators, which are biosynthesized via enzymatic pathways involving lipoxygenase, cyclooxygenase or cytochrome P450 monooxygenase and specifically regulate inflammation and resolution pathways. OBJECTIVES: This study aimed to establish the baseline patterns of PUFAs and oxylipins in AH and TF by their comprehensive lipidomic identification and profiling in humans in the absence of ocular inflammation and comparatively analyze these compounds in the eye liquids of rabbits, the species often employed in investigative ophthalmology. METHODS: Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used for qualitative and quantitative characterization of lipid compounds in the analyzed samples. RESULTS: A total of 28 lipid compounds were identified, including phospholipid derivatives and PUFAs, as well as 22 oxylipins. Whereas the PUFAs included arachidonic, docosahexaenoic and eicosapentaenoic acids, the oxylipins were derived mainly from arachidonic, linoleic and α-linolenic acids. Remarkably, although the concentration of oxylipins in AH was lower compared to TF, these liquids showed pronounced similarity in their lipid profiles, which additionally exhibited noticeable interspecies concordance. CONCLUSION: The revealed correlations confirm the feasibility of rabbit models for investigating pathogenesis and trialing therapies of human eye disorders. The identified metabolite patterns suggest enzymatic mechanisms of oxylipin generation in AH and TF and might be used as a reference in ocular inflammation studies.

Mechanisms and Treatment of Light-Induced Retinal Degeneration-Associated Inflammation: Insights from Biochemical Profiling of the Aqueous Humor.

Ocular inflammation contributes to the pathogenesis of blind-causing retinal degenerative diseases, such as age-related macular degeneration (AMD) or photic maculopathy. Here, we report on inflammatory mechanisms that are associated with retinal degeneration induced by bright visible light, which were revealed while using a rabbit model. Histologically and electrophysiologically noticeable degeneration of the retina is preceded and accompanied by oxidative stress and inflammation, as evidenced by granulocyte infiltration and edema in this tissue, as well as the upregulation of total protein, pro-inflammatory cytokines, and oxidative stress markers in aqueous humor (AH). Consistently, quantitative lipidomic studies of AH elucidated increase in the concentration of arachidonic (AA) and docosahexaenoic (DHA) acids and lyso-platelet activating factor (lyso-PAF), together with pronounced oxidative and inflammatory alterations in content of lipid mediators oxylipins. These alterations include long-term elevation of prostaglandins, which are synthesized from AA via cyclooxygenase-dependent pathways, as well as a short burst of linoleic acid derivatives that can be produced by both enzymatic and non-enzymatic free radical-dependent mechanisms. The upregulation of all oxylipins is inhibited by the premedication of the eyes while using mitochondria-targeted antioxidant SkQ1, whereas the accumulation of prostaglandins and lyso-PAF can be specifically suppressed by topical treatment with cyclooxygenase inhibitor Nepafenac. Interestingly, the most prominent antioxidant and anti-inflammatory benefits and overall retinal protective effects are achieved by simultaneous administrating of both drugs indicating their synergistic action. Taken together, these findings provide a rationale for using a combination of mitochondria-targeted antioxidant and cyclooxygenase inhibitor for the treatment of inflammatory components of retinal degenerative diseases.

Deuterated Arachidonic Acids Library for Regulation of Inflammation and Controlled Synthesis of Eicosanoids: An In Vitro Study.

The synthesis of signal lipids, including eicosanoids, is not fully understood, although it is key to the modulation of various inflammatory states. Recently, isotopologues of essential polyunsaturated fatty acids (PUFAs) deuterated at bis-allylic positions (D-PUFAs) have been proposed as inhibitors of non-enzymatic lipid peroxidation (LPO) in various disease models. Arachidonic acid (AA, 20:4 n-6) is the main precursor to several classes of eicosanoids, which are produced by cyclooxygenases (COX) and lipoxygenases (LOX). In this study we analyzed the relative activity of human recombinant enzymes COX-2, 5-LOX, and 15-LOX-2 using a library of arachidonic acids variably deuterated at the bis-allylic (C7, C10, and C13) positions. Kinetic parameters (KM, Vmax) and isotope effects calculated from kH/kD for seven deuterated arachidonic acid derivatives were obtained. Spectroscopic methods have shown that deuteration at the 13th position dramatically affects the kinetic parameters of COX-2 and 15-LOX-2. The activity of 5-LOX was evaluated by measuring hydroxyeicosatetraenoic acids (8-HETE and 5-HETE) using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Deuteration at the seventh and 10th positions affects the performance of the 5-LOX enzyme. A flowchart is proposed suggesting how to modulate the synthesis of selected eicosanoids using the library of deuterated isotopologues to potentially fine-tune various inflammation stages.