Detection and quantification of endocannabinoids by LC-MS/MS
Final Report Abstract
Translation of (pre)clinical features of a particular disease, as well as of the molecular plasticity underlying physiological and pathophysiological events into quantitative biology is key to expedite the discovery of new drug targets, drug candidates and biomarkers. Within the Central Project 1 of FOR926, we focused primarily on the development of quantitative assays, based on liquid-liquid extraction combined with liquidchromatography multiple reaction monitoring for investigation of endocannabinoids (eCBs), eicosanoids (eiCs), and membrane lipids in various tissues and biological fluids. Additionally, lipid profiling, e.g. nontargeted lipid profiling, lipid class-specific profiling, fatty-acyl content profiling of a lipidome along with relative quantification were developed and implemented as “hypothesis generation” tool for lipids association with various physiological and pathophysiological states. We have set and standardized sampling protocols for cell populations, tissues and biological fluids to minimize the ex-vivo alterations of endogenous levels of lipids. Similarly, lipid extraction and quantification protocols were tailored and standardized to allow intra- and inter-study, as well as inter-laboratory reproducibility. For eCB determinations, standard operating procedures (SOP) for low- and high-throughput quantification, tailored to various tissues types and amounts, and biological fluids were set and implemented in laboratory. The following lipidomic solutions were developed within the Central Project 1 of FOR926: a) Endocannabinoid quantification at low- and high-throughput in a) all brain regions; b) peripheral tissues originating from liver, spleen, pancreas, kidney, lungs, heart, muscle, bones, white and brown fat; c) biological fluids, such as plasma, serum, cerebrospinal fluid, and cell supernatant, and d) various cell types. Determination at increased spatial resolution in brain has succeeded to the extent of brain punches, such as dorsal and ventral hippocampus, thalamus, amygdala, etc. Such determinations were applied and valorized in several research projects including cardiovascular, metabolic, pulmonary and psychological disorders such as stress, anxiety, as well as in aging research. b) Multiplex quantification of endocannabinoids and eicosanoids in tissues and biological fluids based on co-extraction of CBs and eiCs and multiplex quantification by LC/MRM. This method was applied for all brain tissues, peripheral tissues, such as lung, heart and spleen, as well as plasma. eCBs and eiCs changes in acute epileptic seizure models, infectious diseases, as well as pulmonary diseases were so far investigated. c) Multiplex quantification of selected phospholipids (more than 200 components) from all brain tissues, peripheral organs: lung, heart, spleen as well as in plasma. This method was applied in epilepsy research, post-traumatic stress disorder and several infectious disease research. d) Dual extraction method for lipidomic and transcriptomic investigation at increased spatial resolution in brain, e.g. brain punches. This method is based on mRNA extraction and concurrent lipid extraction from a single tissue source, e.g. brain punch, allowing for both targeted phospholipid and eCB quantification and mRNA quantification. This method was applied in epilepsy research. Additionally, the method is suitable also for lower spatial resolution, where bias due to tissue heterogeneity and/or variability in experimental models, otherwise occurring when multi molecular investigations are carried out, can be prevented. The latter method was applied in post-traumatic stress disorder research.
Publications
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(2013) Endocannabinoid anandamide mediates hypoxic pulmonary vasoconstriction. Proc Natl Acad Sci USA 110(46):18710-5
Wenzel D, Matthey M, Bindila L, Lerner R, Lutz B, Zimmer A, Fleischmann BK
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(2014) O-2050 facilitates noradrenaline release and increases the CB1 receptor inverse agonistic effect of rimonabant in the guinea pig hippocampus. Naunyn Schmiedebergs Arch Pharmacol 387(7):621-8
Jergas B, Schulte K, Bindila L, Lutz B, Schlicker E
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(2015) Activation of Endocannabinoid System Is Associated with Persistent Inflammation in Human Aortic Aneurysm. Biomed Res Int 2015:456582
Gestrich C, Duerr GD, Heinemann JC, Meertz A, Probst C, Roell W, Schiller W, Zimmer A, Bindila L, Lutz B, Welz A, Dewald O
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(2015) Age-related changes in the endocannabinoid system in the mouse hippocampus. Mech Ageing Dev 150:55-64
Piyanova A, Lomazzo E, Bindila L, Lerner R, Albayram O, Ruhl T, Lutz B, Zimmer A, Bilkei-Gorzo A
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(2015) Anxiety, Stress, and Fear Response in Mice with Reduced Endocannabinoid Levels. Biol Psychiatry Biol Psychiatry 79:858-68
Jenniches I, Ternes S, Albayram O, Otte DM, Bach K, Bindila L, Michel K, Lutz B, Bilkei-Gorzo A, Zimmer A
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(2015) Impaired border zone formation and adverse remodeling after reperfused myocardial infarction in cannabinoid CB2 receptor deficient mice. Life Sci 138:8-17
Duerr GD, Heinemann JC, Gestrich C, Heuft T, Klaas T, Keppel K, Roell W, Klein A, Zimmer A, Velten M, Kilic A, Bindila L, Lutz B, Dewald O
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(2015) Therapeutic potential of inhibitors of endocannabinoid degradation for the treatment of stress-related hyperalgesia in an animal model of chronic pain. Neuropsychopharmacology 40(2):488-501
Lomazzo E, Bindila L, Remmers F, Lerner R, Schwitter C, Hoheisel U, Lutz B
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(2016) Extraction and Simultaneous Quantification of Endocannabinoids and Endocannabinoid-Like Lipids in Biological Tissues. Methods Mol Biol 1412:9-18
Bindila L, Lutz B
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(2016) Lack of hippocampal CB1 receptor desensitization by Δ9-tetrahydrocannabinol in aged mice and by low doses of JZL 184. Naunyn Schmiedebergs Arch Pharmacol 389:603-12
Feliszek M, Bindila L, Lutz B, Zimmer A, Bilkei-Gorzo A, Schlicker E
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(2016) Myeloid-Specific Deletion of Diacylglycerol Lipase α Inhibits Atherogenesis in ApoE- Deficient Mice. PLoS One 11(1):e0146267
Jehle J, Hoyer FF, Schöne B, Pfeifer P, Schild K, Jenniches I, Bindila L, Lutz B, Lütjohann D, Zimmer A, Nickenig G
