Application of Mass Spectrometry in Unravelling the Secrets of the Endocannabinoid System

The endocannabinoid (EC) system is a regulatory signaling system that is linked to a wide spectrum of physiological and pathophysiological states, such as the modulation of neural development, immune functions, metabolism, food intake, pain perception, cognition, emotion/motivation, psychomotor control, and synaptic plasticity. Such a diverse signaling system has raised high expectations as a target for drug development strategies for the treatment of a wide variety of ailments. In addition EC signaling is linked to several neuropsychiatric disorders that include drug addictions and neurodegenerative diseases. During the last thirty years of research and discovery, many components of the EC system are now well known, yet a complete understanding of this signaling system is lacking. To reveal these remaining scientific mysteries, highly sophisticated analytical techniques are required, in concert with pharmacological and biological strategies. In this study, we identified significant variations in EC levels in specific regions of postmortem alcoholics’ brains, which also correlated with other neurotransmitter systems from the same set of samples. Further, we were the first laboratory to identify the two main endocannabinoids; i.e., N-arachidonoyl ethanolamine and 2-arachidonoylglycerol, in Caenorhabditis elegans (C. elegans), a free living soil nematode that is widely used as a model organism in biological research. We also demonstrated significant changes in the EC levels and global metabolome of these animals after food deprivation and alcohol administration. These findings were based on novel mass spectrometric methods that were developed and validated in our laboratory. We used liquid-liquid extraction to quench the metabolic processes and extract analytes of interest from the biological samples. Linear ion trap and quadrupole time-of-flight detectors were used for qualitative analysis and metabolomics, respectively. The quantitative analysis of ECs was made with a triple quadrupole instrument. Positive electrospray ionization was used in the analysis of ECs. To investigate the metabolomics, we expanded the coverage of metabolites in these samples by using two different liquid chromatographic techniques (i.e., reversed phase and hydrophilic interaction chromatography), in concert with both polarities in electrospray ionization. The results demonstrate mass spectrometry to be a powerful analytical tool for unravelling the secrets of EC system. Furthermore, C. elegans offers a unique, non-CB1R/CB2R-mediated model for the study of EC signaling in vivo. This model will help further our understanding of other physiological roles within the EC system (e.g., distinct physiological roles of Narachidonoylethanolamine and 2-arachidonoylglycerol). National Library of Medicine Classification: QU 25, QU 85, QU 85.6, QU 120, QV 25, QV 126 Medical Subject Headings: Chemistry Techniques, Analytical; Chromatography, Liquid; Mass Spectrometry; Spectrometry, Mass, Electrospray Ionization; Metabolism; Metabolomics; Metabolome; Lipids; Endocannabinoids/analysis; Arachidonic Acids/analysis; Food Deprivation; Ethanol; Brain; Nematoda; Caenorhabditis elegans; Models, Animal