Lipidomic Analysis of Endocannabinoid Signaling: Targeted Metabolite Identification and Quantification

Abstract

The endocannabinoids N-arachidonoylethanolamide (or anandamide, AEA) and 2-arachidonoylglycerol (2-AG) belong to the larger groups of N-acylethanolamines (NAEs) and monoacylglycerol (MAG) lipid classes, respectively. They are biologically active lipid molecules that activate G-protein-coupled cannabinoid receptors found in various organisms. After AEA and 2-AG were discovered in the 1990s, they have been extensively documented to have a broad range of physiological functions. Along with AEA, several NAEs, for example, N-palmitoylethanolamine (PEA), N-stearoylethanolamine (SEA), and N-oleoylethanolamine (OEA) are also present in tissues, usually at much larger concentrations than AEA. Any perturbation that involves the endocannabinoid pathway may subsequently alter basal level or metabolism of these lipid mediators. Further, the altered levels of these molecules often reflect pathological conditions associated with tissue damage. Robust and sensitive methodologies to analyze these lipid mediators are essential to understanding how they act as endocannabinoids. The recent advances in mass spectrometry allow researchers to develop lipidomics approaches and several methodologies have been proposed to quantify endocannabinoids in various biological systems.

Figure 1

Structures of AEA and 2-AG glycerol, the two best known endocannabinoids.

Figure 2

Chemical structures of different classes of endocannabinoids and endocannabinoid-like molecules reported. (a) N-Acylethanolamine, (b) monoacylglycerol, (c) N-acyldopamine, (d) N-acyl amino acid, (e) N-acylamine, (f) N-acylserotonin, (g) COX-2 derivatives, (h) CYP450 derivatives, (i) 15-LOX derivatives, (j) and 12-LOX derivatives.

Figure 3

Commercially available internal standards for analysis by mass spectrometry. (a) Arachidonoyl ethanolamide-d₄ (AEA-d₄). (b) Arachidonoyl ethanolamide-d₈ (AEA-d₈). (c) 2-Arachidonoyl glycerol-d₅ (2-AG-d₅). (d) 2-Arachidonoyl glycerol-d₈ (2-AG-d₈).

Figure 4

Structures of common derivatizing agents used in GC-based analysis. (a) N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA). (b) N-Methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA). (c) tert-Butyl dimethylsilyl (tBDMS). (d) Pentafluorobenzoyl chloride (PFBzCl). (e) Acetic anhydride.

Figure 5

Structures of common derivatizing agents used in HPLC with UV or fluorescence detectors. (a) Dansyl chloride. (B) 4-(N-Chloroformylmethyl-N-methyl) amino-7-N,N-dimethylaminosulphonyl-2,1,3-benzoxadiazole (DBD-COCl).

Figure 6

Fragmentations from LC/MS/MS in positive ESI mode of (a) [AEA + H]⁺ at m/z 348.4 and (b) [2-AG + H]⁺ at m/z 379.2 after CID [19].

Figure 7

Mass spectra of NAEs generated by GC-MS with positive EI in scan mode. (a) N-Palmitoylethanolamine (PEA). (b) N-Stearoylethanolamine (SEA). (c) N-Arachidonoylethanolamide (AEA). (Left column represents authentic, purified compounds; right column represents the same compounds identified in retinal tissue extracts.)