Analysis of Omega-3 Fatty Acid-Derived N-Acylethanolamines in Biological Matrices.

The adequate quantification of endocannabinoids and related N-acylethanolamines can be complex due to their low endogenous levels, structural diversity, and metabolism. Therefore, advanced analytical approaches, involving LC-MS, are required to quantify these molecules in plasma, tissues, and other matrices. It has been shown that endocannabinoid congeners synthesized from n-3 poly-unsaturated fatty acids (n-3 PUFAs), such as docosahexaenoylethanolamide (DHEA) and eicosapentaenoylethanolamide (EPEA), have interesting immunomodulatory and tumor-inhibiting properties. Recent work has shown that DHEA and EPEA can be further enzymatically metabolized by cyclo-oxygenase 2 (COX-2), forming oxygenated metabolites. Here, an LC-MS-based method for the quantification of the n-3 PUFA-derived endocannabinoid congeners DHEA and EPEA is described, which is also suited to measure a wider spectrum of endocannabinoids. The chapter contains a step-by-step protocol for the analysis of (n-3) endocannabinoids in plasma, including sample collection and solid phase extraction, LC-MS analysis, and data processing. In addition, protocol modifications are provided to allow quantification of n-3 PUFA-derived endocannabinoids and their COX-2 metabolites in tissues and cell culture media. Finally, conditions that alter endocannabinoid concentrations are briefly discussed.

PUFA-Derived N-Acylethanolamide Probes Identify Peroxiredoxins and Small GTPases as Molecular Targets in LPS-Stimulated RAW264.7 Macrophages.

We studied the mechanistic and biological origins of anti-inflammatory poly-unsaturated fatty acid-derived N-acylethanolamines using synthetic bifunctional chemical probes of docosahexaenoyl ethanolamide (DHEA) and arachidonoyl ethanolamide (AEA) in RAW264.7 macrophages stimulated with 1.0 µg mL-1 lipopolysaccharide. Using a photoreactive diazirine, probes were covalently attached to their target proteins, which were further studied by introducing a fluorescent probe or biotin-based affinity purification. Fluorescence confocal microscopy showed DHEA and AEA probes localized in cytosol, specifically in structures that point toward the endoplasmic reticulum and in membrane vesicles. Affinity purification followed by proteomic analysis revealed peroxiredoxin-1 (Prdx1) as the most significant binding interactor of both DHEA and AEA probes. In addition, Prdx4, endosomal related proteins, small GTPase signaling proteins, and prostaglandin synthase 2 (Ptgs2, also known as cyclooxygenase 2 or COX-2) were identified. Lastly, confocal fluorescence microscopy revealed the colocalization of Ptgs2 and Rac1 with DHEA and AEA probes. These data identified new molecular targets suggesting that DHEA and AEA may be involved in reactive oxidation species regulation, cell migration, cytoskeletal remodeling, and endosomal trafficking and support endocytosis as an uptake mechanism.

Immunomodulating effects of 13- and 16-hydroxylated docosahexaenoyl ethanolamide in LPS stimulated RAW264.7 macrophages.

Docosahexaenoyl ethanolamide (DHEA), the ethanolamine conjugate of the n-3 long chain polyunsaturated fatty acid docosahexaenoic acid, is endogenously present in the human circulation and in tissues. Its immunomodulating properties have been (partly) attributed to an interaction with the cyclooxygenase-2 (COX-2) enzyme. Recently, we discovered that COX-2 converts DHEA into two oxygenated metabolites, 13- and 16-hydroxylated-DHEA (13- and 16-HDHEA, respectively). It remained unclear whether these oxygenated metabolites also display immunomodulating properties like their parent DHEA. In the current study we investigated the immunomodulating properties of 13- and 16-HDHEA in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. The compounds reduced production of tumor necrosis factor alpha (TNFα), interleukin (IL)-1ß and IL-1Ra, but did not affect nitric oxide (NO) and IL-6 release. Transcriptome analysis showed that the compounds inhibited the LPS-mediated induction of pro-inflammatory genes (InhbA, Ifit1) and suggested potential inhibition of regulators such as toll-like receptor 4 (TLR4), MyD88, and interferon regulatory factor 3 (IRF3), whereas anti-inflammatory genes (SerpinB2) and potential regulators IL-10, sirtuin 1 (Sirt-1), fluticasone propionate were induced. Additionally, transcriptome analysis of 13-HDHEA suggests a potential anti-angiogenic role. In contrast to the known oxylipin-lowering effects of DHEA, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analyses revealed that 13- and 16-HDHEA did not affect oxylipin formation. Overall, the anti-inflammatory effects of 13-HDHEA and 16-HDHEA are less pronounced compared to their parent molecule DHEA. Therefore, we propose that COX-2 metabolism of DHEA acts as a regulatory mechanism to limit the anti-inflammatory properties of DHEA.

Novel COX-2 products of n-3 polyunsaturated fatty acid-ethanolamine-conjugates identified in RAW264.7 macrophages.

Cyclooxygenase 2 (COX-2) plays a key role in the regulation of inflammation by catalyzing the oxygenation of PUFAs to prostaglandins (PGs) and hydroperoxides. Next to this, COX-2 can metabolize neutral lipids, including endocannabinoid-like esters and amides. We developed an LC-HRMS-based human recombinant (h)COX-2 screening assay to examine its ability to also convert n-3 PUFA-derived N-acylethanolamines. Our assay yields known hCOX-2-derived products from established PUFAs and anandamide. Subsequently, we proved that eicosapentaenoylethanolamide (EPEA), the N-acylethanolamine derivative of EPA, is converted into PGE3-ethanolamide (PGE3-EA), and into 11-, 14-, and 18-hydroxyeicosapentaenoyl-EA (11-, 14-, and 18-HEPE-EA, respectively). Interestingly, we demonstrated that docosahexaenoylethanolamide (DHEA) is converted by hCOX-2 into the previously unknown metabolites, 13- and 16-hydroxy-DHEA (13- and 16-HDHEA, respectively). These products were also produced by lipopolysaccharide-stimulated RAW267.4 macrophages incubated with DHEA. No oxygenated DHEA metabolites were detected when the selective COX-2 inhibitor, celecoxib, was added to the cells, further underlining the role of COX-2 in the formation of the novel hydroxylated products. This work demonstrates for the first time that DHEA and EPEA are converted by COX-2 into previously unknown hydroxylated metabolites and invites future studies toward the biological effects of these metabolites.

The role of n-3 PUFA-derived fatty acid derivatives and their oxygenated metabolites in the modulation of inflammation.

Notwithstanding the ongoing debate on their full potential in health and disease, there is general consensus that n-3 PUFAs play important physiological roles. Increasing dietary n-3 PUFA intake results in increased DHA and EPA content in cell membranes as well as an increase in n-3 derived oxylipin and -endocannabinoid concentrations, like fatty acid amides and glycerol-esters. These shifts are believed to (partly) explain the pharmacological and anti-inflammatory effects of n-3 PUFAs. Recent studies discovered that n-3 PUFA-derived endocannabinoids can be further metabolized by the oxidative enzymes CYP-450, LOX and COX, similar to the n-6 derived endocannabinoids. Interestingly, these oxidized n-3 PUFA derived endocannabinoids of eicosapentaenoyl ethanolamide (EPEA) and docosahexaenoyl ethanolamide (DHEA) have higher anti-inflammatory and anti-proliferative potential than their precursors. In this review, an overview of recently discovered n-3 PUFA derived endocannabinoids and their metabolites is provided. In addition, the use of chemical probes will be presented as a promising technique to study the n-3 PUFA and n-3 PUFA metabolism within the field of lipid biochemistry.

Highly Specific Binding on Antifouling Zwitterionic Polymer-Coated Microbeads as Measured by Flow Cytometry.

Micron- and nano-sized particles are extensively used in various biomedical applications. However, their performance is often drastically hampered by the nonspecific adsorption of biomolecules, a process called biofouling, which can cause false-positive and false-negative outcomes in diagnostic tests. Although antifouling coatings have been extensively studied on flat surfaces, their use on micro- and nanoparticles remains largely unexplored, despite the widespread experimental (specifically, clinical) uncertainties that arise because of biofouling. Here, we describe the preparation of magnetic micron-sized beads coated with zwitterionic sulfobetaine polymer brushes that display strong antifouling characteristics. These coated beads can then be equipped with recognition elements of choice, to enable the specific binding of target molecules. First, we present a proof of principle with biotin-functionalized beads that are able to specifically bind fluorescently labeled streptavidin from a complex mixture of serum proteins. Moreover, we show the versatility of the method by demonstrating that it is also possible to functionalize the beads with mannose moieties to specifically bind the carbohydrate-binding protein concanavalin A. Flow cytometry was used to show that thus-modified beads only bind specifically targeted proteins, with minimal/near-zero nonspecific protein adsorption from other proteins that are present. These antifouling zwitterionic polymer-coated beads, therefore, provide a significant advancement for the many bead-based diagnostic and other biosensing applications that require stringent antifouling conditions.