Abstract
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.
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References
Pertwee RG, Howlett AC, Abood ME, Alexander SPH, Di Marzo V, Elphick MR et al (2010) International Union of Basic and Clinical Pharmacology. LXXIX. Cannabinoid receptors and their ligands: beyond CB1 and CB2. Pharmacol Rev 62:588–631
Pertwee RG (2015) Endocannabinoids and their pharmacological actions. In: Pertwee RG (ed) Endocannabinoids. Springer, Cham, pp 1–37
Artmann A, Petersen G, Hellgren LI, Boberg J, Skonberg C, Nellemann C et al (2008) Influence of dietary fatty acids on endocannabinoid and N-acylethanolamine levels in rat brain, liver and small intestine. Biochimica et Biophysica Acta (BBA) – Mol Cell Biol Lipids 1781:200–212
Matias I, Carta G, Murru E, Petrosino S, Banni S, Di Marzo V (2008) Effect of polyunsaturated fatty acids on endocannabinoid and N-acyl-ethanolamine levels in mouse adipocytes. Biochimica et Biophysica Acta (BBA) – Mol Cell Biol Lipids 1781:52–60
Balvers M, Verhoeckx K, Bijlsma S, Rubingh C, Meijerink J, Wortelboer H et al (2012) Fish oil and inflammatory status alter the n-3 to n-6 balance of the endocannabinoid and oxylipin metabolomes in mouse plasma and tissues. Metabolomics 8:1130–1147
Balvers MGJ, Verhoeckx KCM, Plastina P, Wortelboer HM, Meijerink J, Witkamp RF (2010) Docosahexaenoic acid and eicosapentaenoic acid are converted by 3T3-L1 adipocytes to N-acyl ethanolamines with anti-inflammatory properties. Biochim Biophys Acta Mol Cell Biol Lipids 1801:1107–1114
Alharthi N, Christensen P, Hourani W, Ortori C, Barrett DA, Bennett AJ et al (2018) n-3 polyunsaturated N-acylethanolamines are CB2 cannabinoid receptor-preferring endocannabinoids. Biochim Biophys Acta Mol Cell Biol Lipids 1863:1433–1440
Bradshaw HB, Raboune S, Hollis JL (2013) Opportunistic activation of TRP receptors by endogenous lipids: exploiting lipidomics to understand TRP receptor cellular communication. Life Sci 92:404–409
Di Marzo V, Bisogno T, De Petrocellis L (2007) Endocannabinoids and related compounds: walking back and forth between plant natural products and animal physiology. Chem Biol 14:741–756
Murru E, Lopes PA, Carta G, Manca C, Abolghasemi A, Guil-Guerrero JL et al (2021) Different dietary N-3 polyunsaturated fatty acid formulations distinctively modify tissue fatty acid and N-acylethanolamine profiles. Nutrients 13:1
Zoerner AA, Gutzki F-M, Batkai S, May M, Rakers C, Engeli S et al (2011) Quantification of endocannabinoids in biological systems by chromatography and mass spectrometry: a comprehensive review from an analytical and biological perspective. Biochimica et Biophysica Acta (BBA) – Mol Cell Biol Lipids 1811:706–723
Balvers MGJ, Wortelboer HM, Witkamp RF, Verhoeckx KCM (2013) Liquid chromatography–tandem mass spectrometry analysis of free and esterified fatty acid N-acyl ethanolamines in plasma and blood cells. Anal Biochem 434:275–283
Meijerink J, Plastina P, Vincken J-P, Poland M, Attya M, Balvers MGJ et al (2011) The ethanolamide metabolite of DHA, docosahexaenoylethanolamine, shows immunomodulating effects in mouse peritoneal and RAW264.7 macrophages: evidence for a new link between fish oil and inflammation. Br J Nutr 105:1789–1807
Meijerink J, Poland M, Balvers MGJ, Plastina P, Lute C, Dwarkasing J et al (2015) Inhibition of COX-2-mediated eicosanoid production plays a major role in the anti-inflammatory effects of the endocannabinoid N-docosahexaenoylethanolamine (DHEA) in macrophages. Br J Pharmacol 172:24–37
Brown I, Cascio MG, Wahle KWJ, Smoum R, Mechoulam R, Ross RA et al (2010) Cannabinoid receptor-dependent and -independent anti-proliferative effects of omega-3 ethanolamides in androgen receptor-positive and -negative prostate cancer cell lines. Carcinogenesis 31:1584–1591
Rovito D, Giordano C, Vizza D, Plastina P, Barone I, Casaburi I et al (2013) Omega-3 PUFA ethanolamides DHEA and EPEA induce autophagy through PPARγ activation in MCF-7 breast cancer cells. J Cell Physiol 228:1314–1322
Kim HY, Spector AA, Xiong ZM (2011) A synaptogenic amide N-docosahexaenoylethanolamide promotes hippocampal development. Prostaglandins Other Lipid Mediat 96:114–120
de Bus I, Witkamp R, Zuilhof H, Albada B, Balvers M (2019) The role of n-3 PUFA-derived fatty acid derivatives and their oxygenated metabolites in the modulation of inflammation. Prostaglandins Other Lipid Mediat 144:106351
de Bus I, Zuilhof H, Witkamp R, Balvers M, Albada B (2019) Novel COX-2 products of n-3 polyunsaturated fatty acid-ethanolamine-conjugates identified in RAW264.7 macrophages. J Lipid Res 60:1829–1840
de Bus I, van Krimpen S, Hooiveld GJ, Boekschoten MV, Poland M, Witkamp RF et al (2021) Immunomodulating effects of 13- and 16-hydroxylated docosahexaenoyl ethanolamide in LPS stimulated RAW264.7 macrophages. Biochim Biophys Acta – Mol Cell Biol Lipids 1866:158908
Balvers MGJ, Verhoeckx KCM, Witkamp RF (2009) Development and validation of a quantitative method for the determination of 12 endocannabinoids and related compounds in human plasma using liquid chromatography-tandem mass spectrometry. J Chromatogr B 877:1583–1590
Balvers MGJ, Verhoeckx KCM, Meijerink J, Bijlsma S, Rubingh CM, Wortelboer HM et al (2012) Time-dependent effect of in vivo inflammation on eicosanoid and endocannabinoid levels in plasma, liver, ileum and adipose tissue in C57BL/6 mice fed a fish-oil diet. Int Immunopharmacol 13:204–214
Joosten M, Balvers M, Verhoeckx K, Hendriks H, Witkamp R (2010) Plasma anandamide and other N-acylethanolamines are correlated with their corresponding free fatty acid levels under both fasting and non-fasting conditions in women. Nutr Metab 7:49
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Witkamp, R.F., de Bus, I., Albada, B., Balvers, M.G.J. (2023). Analysis of Omega-3 Fatty Acid-Derived N-Acylethanolamines in Biological Matrices. In: Maccarrone, M. (eds) Endocannabinoid Signaling. Methods in Molecular Biology, vol 2576. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2728-0_5
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DOI: https://doi.org/10.1007/978-1-0716-2728-0_5
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