Identification of endocannabinoids and related N-acylethanolamines in tetrahymena. A new class of compounds for Tetrahymena.

The endocannabinoid system is a lipid signaling system in mammalian cells. We reported that major components of the endocannabinoid system such as fatty acid amide hydrolase and monoacylglycerol lipase, are present in the protist Tetrahymena, with characteristics similar to those in mammals. Tetrahymena is a model organism for molecular and cellular biology studies as its genome sequence is available. Here we report the presence of N-acylethanolamines (AcEs) and their respective 2-acylglycerols (2-AcGs) in Tetrahymena thermophila for the first time; the former is a new lipid class for the protist. Using LC-MS/MS we identified, N y-linolenoyl, N-eicosenoyl, N-linoleoyl, N-palmitoyl, N-stearoyl and N-oleoylethanolamines as well as the corresponding monoacylglycerols. The levels of 2-acylglycerols were much higher than the corresponding N-acylethanolamines, as reported for mammals. To our knowledge, N-gamma-linolenoylethanolamine (GLEA) was found for the first time in nature. Anandamide and 2-AG were present in trace amounts. These results demonstrate the existence of a new lipid class in Tetrahymena, strengthen the conviction that the endocannabinoid system is present in this protist, verifying its importance throughout evolution. Tetrahymena could be used as a model for metabolic studies on the endocannabinoids, as well as for the study of drugs targeted towards biosynthetic and catabolic enzymes of AcEs and 2-AcGs.

Comprehensive profiling of the human circulating endocannabinoid metabolome: clinical sampling and sample storage parameters.

BACKGROUND: Endogenous cannabinoid-receptor ligands (endocannabinoids) and over a dozen related metabolites now comprise the "endocannabinoid metabolome". The diverse (patho)physiological roles of endocannabinoids, the predictive/diagnostic utility of systemic endocannabinoid levels, and the growing interest in endocannabinoid-related pharmacotherapeutics mandate a valid clinical protocol for processing human blood that does not jeopardize profiling of the circulating endocannabinoid metabolome. METHODS: We systematically evaluated the potential effect of pre-analytical variables associated with phlebotomy and sample handling/work-up on the human-blood endocannabinoid metabolome as quantified by state-of-the-art liquid chromatography-mass spectrometry. RESULTS: Neither subject posture during phlebotomy nor moderate activity beforehand influenced the blood levels of the 15 endocannabinoid-system lipids quantified. Storage of fresh blood at 4 degrees C selectively enhanced ethanolamide concentrations artifactually without affecting monoglycerides and nonesterified fatty acids, such as arachidonic acid. In marked contrast, ethanolamides and monoglycerides remained stable through three plasma freeze/thaw cycles, whereas plasma arachidonic acid content increased, probably a reflection of ongoing metabolism. CONCLUSIONS: Class- and compound-selective pre-analytical influences on circulating human endocannabinoid levels necessitate immediate plasma preparation from fresh blood and prompt plasma apportioning and snap-freezing. Repeated plasma thawing and refreezing should be avoided. This protocol ensures sample integrity for evaluating the circulating endocannabinoid metabolome in the clinical setting.