Release of anandamide from blood cells

Abstract

Background: Endogenous ligands of cannabinoid receptors (endocannabinoids), in particular anandamide (arachidonylethanolamide), have been recognized as being of crucial importance in a variety of physiological functions. Plasma concentrations of anandamide have been measured in a number of investigations; however, discrepant data on ''normal'' anandamide plasma concentrations were reported. Since this might be caused by pre-analytical variables, we investigated the impact of different sample handling conditions on measured plasma anandamide concentrations. Methods: Blood samples were taken from healthy volunteers in EDTA-or heparin-containing tubes; whole blood samples were kept at q48C, room temperature, or 378C, respectively, for up to 120 min before obtaining plasma by centrifugation. Plasma anandamide concentrations were measured by an isotope-dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Results: A marked time-and temperature-dependent increase in plasma anandamide concentrations ex vivo was observed in both EDTA-and heparin-containing tubes. Mean anandamide concentrations approximately doubled when EDTA samples were kept at 48C for 60 min before centrifugation wimme-

A solid-phase method for the extraction and measurement of anandamide from multiple human biomatrices

“…The levels of AEA measured by both LPE and SPE methods in plasma from non-pregnant woman was 0.64 nM and 1.38 nM (range 0.51-3.99 nM) for term pregnant women. These data differ from those reported by other groups, 2.58 and 3.73 nM for non-pregnant women [23,24]. The difference could be related to the physiological state or the timing of processing of their biological samples.…”

“…Furthermore, it has also been demonstrated that use of silica SPE, but not C18 SPE, can lead to loss of deuterium from internal standard [21] causing inaccuracy in the determination of AEA. In addition, these methods were developed for brain tissue using concentrations of AEA ranging from 20-2000 pmol/g [10,11,21] which are not representative of the low pmol/ml AEA concentrations observed in plasma [12,13,16,17,24]. An additional problem when modifying AEA extraction methods for tissues for use with bio-fluids is that extraction media are added to tissues in large quantities (40-500 fold w/v) [10,11,21] which is possible because of the small size of the tissue samples being extracted.…”

Dysregulation of the Peripheral and Adipose Tissue Endocannabinoid System in Human Abdominal Obesity

“…Several aspects, e.g., clinical condition, concomitant medication, fasting/nonfasting state, and possibly also sex differences, need to be considered. Furthermore, some methodological precautions must be followed to avoid artificially increased anandamide levels in blood (34).…”

Ultra performance liquid chromatography tandem mass spectrometry method for the measurement of anandamide in human plasma

“…Measurements of endocannabinoids by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) in three recent studies in rat brain and human plasma have reduced analysis times to between 7-15 min. However, these studies have either a: not been validated [18], b: are used for brain tissues not plasma [19] or c: had comparatively poor limits of detection [20].…”

“…Although plasma AEA concentrations have been determined in a number of human cohorts, the mean AEA concentrations reported have varied considerably between studies, from 0.37 nM [18] and 2.58 nM [21] to 3.73 nM [20]. This 10-fold variability between studies suggests that plasma AEA concentrations can be difficult to determine routinely and thus a robust, validated analysis method is needed.…”