Lipidomic analysis of endocannabinoid metabolism in biological samples

Abstract

The endocannabinoids are signaling lipids present in many living organisms. They activate G protein-coupled cannabinoid receptors to modulate a broad range of biological processes that include emotion, cognition, inflammation and reproduction. The endocannabinoids are embedded in an interconnected network of cellular lipid pathways, the regulation of which is likely to control the strength and duration of endocannabinoid signals. Therefore, physiopathological or pharmacological perturbations of these pathways may indirectly affect endocannabinoid activity and, vice versa, endocannabinoid activity may influence lipid pathways involved in other metabolic and signaling events. Recent progress in liquid chromatography and mass spectrometry has fueled the development of targeted lipidomic approaches, which allow researchers to examine complex lipid interactions in cells and gain a broader view of the endocannabinoid system. Here, we review these new developments from the perspective of our laboratory's experience in the field.

Figure 1. Lipidomic analysis of the endocannabinoid metabolism

Flow chart of the strategy used to profile endocannabinoid lipids from biological samples. Abbreviations: DAG, diacylglycerol; FA, fatty acid; FAE, fatty acid ethanolamide; MAG, monoacylglycerol; NAPE, N-acyl-phosphatidylethanolamine; LNAPE, lyso-NAPE; oxFA, oxygenated fatty acids; oxFAE, oxygenated FAE; oxMAG, oxygenated MAG; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; TAG, triacylglycerol.

Figure 2. LC/MS metabolite discovery tools

A, Representative total ion LC/MS chromatogram of a mixture of isobaric and isomeric N-acyl phosphatidylethanolamine (NAPE) species (top). A 3D LC/MS contour mapping (bottom) visualizes individual molecular species present in the mixture. The first dimension is elution time, the second is mass-to-charge ratio (m/z) and the third relative intensity of the signal, which is symbolized with a pseudocolor scale. LC/MSⁿ conditions are those previously reported [12]. Three-dimensional maps were generated using MS Processor from Advanced Chemistry Development, Inc. (Toronto, Canada). B, Representative ESI/MSⁿ analysis of N-arachidonoyl phosphoethanolamide species in the rat brain. MS² and MS³ lead to fragment ions that are found physiologically as neutral molecular species: lyso-NAPE (LNAPE), phosphoanandamide (PAEA), oleic acid (OA), stearic aldehyde (SAd), cyclic-lysophosphatidic acid (cLPA) and a cyclic glycerophospho-anandamide (cGPAEA, which has not yet been identified in mammals).

Figure 3. Anandamide metabolism: an overview

Postulated pathways of anandamide metabolism. Abbreviations: PC, phosphatidylcholine; PE, phosphatidylethanolamine; NAT, N-acyl transferase; LPA, lysophosphatidic acid; PA, phosphatidic acid; NAPE, N-acyl-phosphatidylethanolamine; LNAPE, 1-lyso,2-acyl-sn-glycero-3-phosphoethanolamine-N-acyl; GP-anadamide, glycerophospho-anandamide; PAEA, phosphoanandamide; PLA, phospholipase A; ABHD, α/β hydrolase; NAPE-PLD, NAPE phospholipase D; PLC, phospholipase C; FAAH, fatty acid amide hydrolase; P, phosphatase; PG, prostaglandin; EET, epoxyeicosatrienoic acid; HETE, hydroxyeicosatetraenoic acid; COX, cyclooxygenase; PGS, prostaglandin synthase; LOX, lipoxygenase; CYP450; cytochrome P450, PDE, phosphodiesterase.

Figure 4. Lipid remodeling and anandamide metabolism: analysis of phosphatidylethanolamine (PE) species

Representative LC/MS analysis of PE species in a human brain. LC/MS chromatogram (top) and MS² fragmentation pattern using an ion trap mass spectrometer (bottom). PE species were detected as deprotonated molecular ions in the negative mode. In MS², the most prominent fragments are the sn-2 lyso-PE in combination with the sn-1 and sn-2 carboxylate anions [90]. Sample characteristics are described in the text. Lipids were extracted from approximately 50 mg of brain tissue and resuspended in 0.1 ml of methanol. Injection volume was 10 μl. Separation was performed on a SB300 Poroshell column (Agilent-Technologies coating layer of 0.25 μm on total particle diameter of 5 μm) using a linear gradient of methanol in water containing 0.25% acetic acid and 5 mM ammonium acetate (from 85% to 100% of methanol in 5 min) at a flow rate of 1.0 ml/min with column temperature set at 50 °C. Capillary voltage was 4.5kV, skim1 -40V, and capillary exit -151V. N₂ was used as drying gas at a flow rate of 12 liters/min, temperature of 350°C and nebulizer pressure of 80 PSI. Helium was used as collision gas. Abbreviations: R₁=sn-1 aliphatic chain; R₂=sn-2 aliphatic chain.

Figure 5. Lipid remodeling and anandamide metabolism: analysis of phosphatidylcholine (PC) species

Identification of 1,2-diarachidonoyl PC in human brain samples. LC/MS chromatogram (top) and fragmentation pattern in MS² and MS³ using an ion trap instrument (bottom). Sample characteristics are described in the text and LC/MS conditions in Fig. 4 legend. Lipids were extracted from approximately 50 mg of brain tissue and resuspended in 0.1 ml of methanol. Injection volume was 10 μl. PC species were detected as acetate adducts of the molecular ions using ESI set in the negative mode. The MS² fragmentation pattern is characterized by neutral loss of the acetate adduct of the N-methyl group. MS³ of the ion with m/z 814.5 yields the lysophospholipid with neutral loss of ketene in combination with the sn-1 and sn-2 carboxylate anions. Abbreviations: R₁=sn-1 aliphatic chain; R₂=sn-2 aliphatic chain.

Figure 6. Anandamide precursors: analysis of NAPE and lyso-NAPE species

Identification of endogenous NAPE (A) and lyso-NAPE species (B) in a human brain sample using LC coupled to an ion trap instrument. Sample characteristics are described in the text and LC/MS conditions in Fig. 4 legend. Lipids were extracted from approximately 50 mg of brain tissue and resuspended in 0.1 ml of methanol. Injection volume was 10 μl. A 3D LC/MS contour mapping (A, top) of lipid extracts visualized the individual NAPE species, which were detected as deprotonated molecular ion in the negative ion mode. The first dimension is the elution time, the second is m/z ratio and the third the relative intensity of the signal, which is represented by a pseudocolor scale. In MS², the product ions are mainly sn-2 lysophospholipid with neutral loss of ketene, the sn-2 carboxylate anion and a series of characteristic minor fragments (A, bottom) [12]. Lyso-NAPE species were separated by PE and NAPE species using the chromatographic conditions described in Fig. 4 legend (B, top). In MS², the lyso-NAPE yielded the putative cyclic glycerophospho-anandamide and sn-1 carboxylate anions (B, bottom) [90].

Figure 7. Anandamide analogs: fatty acid ethanolamides (FAEs)

LC/MSⁿ analysis of endogenous cannabimimetic and non-cannabimimetic fatty acid ethanolamides (FAEs) in human brain. Sample characteristics are described in the text. Lipids were extracted from approximately 50 mg of brain tissue and resuspended in 0.1 ml of methanol. Injection volume was 10 μl. FAEs were detected as sodium adducts of the molecular ions using ESI set in the positive mode. LC/MS conditions are as described [64].

Figure 8. 2-AG metabolism: an overview

Postulated pathways for 2-AG metabolism. Abbreviations: PLC, phospholipase C; DAG, diacylglycerol; DGL, diacylglycerol lipase; MAG, monoacylglycerol; MGL, monoacylglycerol lipase; PLA, phospholipase A; AT, acyltransferase; TAGL, triacylglycerol lipase; PIP₂, phosphatidylinositol bisphosphate; ABDH, α/β hydrolase; lyso-PL, lysophospholipid; lyso-PA, lysophosphatidic acid; PA, phosphatidic acid; P, phosphatase; PG-GE, prostaglandin glycerol ester; PG, prostaglandin; EET-GE, epoxyeicosatrienoyl glycerol ester; EET, epoxyeicosatrienoi acid; HETE-GE, hydroxyeicosatetraenoyl glycerol ester; COX, cyclooxygenase; PGS, prostaglandin synthase; LOX, lipoxygenase; CYP450, cytochrome P450; CDP, cytidine diphosphate.

Figure 9. Lipid remodeling for 2-AG: analysis of phosphatidylinositol (PI) and phosphatidylcholine (PC) species

A, Representative LC/MSⁿ analysis of 1-stearoyl,2-arachidonoyl-sn-glycero-3-phosphoinositol in a human brain. Sample characteristics are described in the text. LC/MS conditions in Fig. 4 legend. Lipids were extracted from approximately 50 mg of brain tissue and resuspended in 0.1 ml of methanol. Injection volume was 10 μl. In the negative ion mode PI was detected as deprotonated molecular ion. MS² yielded the sn-2 lyso-PI together with the sn-1 and sn-2 carboxylate anions. Furthermore, the neutral loss of a ketene in combination with the neutral loss of the inositol head group yielded a putative cyclic sn-1 lysophosphatidic acid derivative. B, Representative LC/MSⁿ analysis of 1-stearoyl,2-arachidonoyl-sn-glycero-3-phosphocholine in a human brain sample. Chromatographic conditions are described in Fig. 4 legend. PC was detected as protonated molecular ion in the positive ion mode. MS² yielded product ions deriving from the neutral loss of the trimethylammonium and the phosphocholine head group. Abbreviations: R₁=sn-1 aliphatic chain; R₂=sn-2 aliphatic chain.

Figure 10. Lipid remodeling and 2-AG metabolism: analysis of triacylglycerols (TAGs)

3D representation of the TAG species present in human brain (left). Proposed structure for the arachidonoyl-containing TAG with m/z 903.9 identified by LC/MS² in human brain tissue (right). LC/MS conditions are described in Fig. 4. Lipids were extracted from approximately 50 mg of brain tissue and resuspended in 0.1 ml of methanol. Injection volume was 1 μl. TAG species were detected as sodium adducts of the molecular ions using ESI set in the positive mode. Using an ion trap instrument, MS² yielded a series of fragments deriving from the neutral loss of the sn-1, sn-2 and sn-3 carboxylate anions. Three-dimensional maps were generated using MS Processor from Advanced Chemistry Development, Inc. (Toronto, Canada). Abbreviations: AA, arachidonic acid; PA, palmitic acid; OA, oleic acid.

Figure 11. 2-AG formation: analysis of DAG and 2-AG

Representative LC/MS analysis of DAG (left) and 2-AG (right) in human brain. Lipids were extracted from approximately 50 mg of brain tissue and resuspended in 0.1 ml of methanol. Injection volume was 10 μl. 2-AG partially isomerizes to 1-AG during extraction and is therefore detected as a double peak [91]. LC/MS conditions are those described [15]. Abbreviations: R₁=sn-1 aliphatic chain; R₂=sn-2 aliphatic chain.