Evaluation of Circadian Variations of Circulating Endocannabinoids

Sarvesh Kumar Verma
University of Florida

Co-authors: Sarvesh Kumar Verma¹, Abhishek Gour¹, Azin Behnood-Rod¹, Barry Setlow¹, John Neubert², Niall Murphy², Abhisheak Sharma^1
1University of Florida, ²Texas A&M University

Background: The endocannabinoid system (ECS) is a lipid signaling network involved in central nervous system development and the regulation of numerous physiological and cognitive functions. It comprises endogenous cannabinoid ligands, cannabinoid receptors, and the enzymes responsible for their synthesis and degradation. Anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are the primary endogenous ligands that activate cannabinoid receptors. However, accurate quantification of these endocannabinoids in blood remains challenging because of their low circulating concentrations, poor stability, and rapid enzymatic degradation following sample collection.

Objective: To develop and validate a robust analytical strategy for stabilization, extraction, and quantification of AEA and 2-AG in blood and to evaluate their blood distribution and 24-hour concentration-time profiles in rats.

Methods: Blood-to-plasma partitioning of AEA and 2-AG was evaluated after 1 h incubation at 37°C by measuring their concentrations in whole blood and plasma. Endocannabinoid stability was then assessed under treated and untreated conditions, with whole blood samples either immediately stabilized with phenylmethylsulfonyl fluoride and acetic acid or left untreated to evaluate degradation. An optimized liquid-liquid extraction method was subsequently developed for the quantitative analysis of AEA and 2-AG in rat whole blood. Finally, the BASi Culex automated sampling system was used to collect serial blood samples over 24 h from female Fischer 344 × Brown Norway F1 hybrid rats to characterize the concentration–time profiles and circadian patterns of circulating AEA and 2-AG.

Results: AEA degraded rapidly under untreated conditions, with only 47.9 ± 8.3% remained after 1 h of incubation, whereas stabilization effectively maintained AEA stability during sample handling. Blood-to-plasma ratio analysis revealed distinct distribution patterns, with ratios of 1.0 ± 0.1 for AEA and 37.1 ± 2.3 for 2-AG, demonstrating preferential partitioning of 2-AG into blood components. Rats exhibited consistently higher circulating 2-AG levels compared to AEA throughout the 24-hour light-dark cycle.

Conclusion: This study presents a reliable analytical method for stabilization, extraction, and quantification of circulating endocannabinoids in blood. Sample stabilization improved endocannabinoid measurement, while blood-to-plasma partitioning analysis demonstrated that whole blood is the preferred matrix for 2-AG quantification. This approach provides a practical approach for investigating endocannabinoid dynamics and evaluating the effects of novel chemical entities targeting the endocannabinoid system.