Quantitative profiling of endocannabinoids in lipoproteins by LC–MS/MS

Abstract

Endocannabinoids belong to a diverse family of endogenous lipid bioregulators acting as physiological ligands of cannabinoid receptor type 1 and cannabinoid receptor type 2 in the central and peripheral nervous system. They are also present in nmol L(-1) concentrations in human blood plasma; however, their association with possible molecular carriers remains poorly characterized. Here we report on the quantification of 46 endogenous molecular species from five major classes of endocannabinoids and endocannabinoid-related compounds in three lipoprotein fractions of human blood plasma: VLDL, LDL, HDL, and in the plasma lipoprotein-free fraction. Although sizable quantities of endocannabinoid-related molecules are associated with lipoproteins, we identified the lipoprotein-free fraction as a major carrier of endocannabinoids in blood circulation with the exception of 2-acylglycerols, which are markedly abundant in VLDL.

Identification of a Widespread Palmitoylethanolamide Contamination in Standard Laboratory Glassware

“…For most compounds, concentrations measured in the present study were in close agreement with those previously reported by our group 6 and others. 7 PEA levels were similar to those reported by Bilgin et al 18 and by Sipe et al, 19 but 10 to 20 times higher than those reported by other groups 7–17 and previously by us. 6 This suggested that a contamination might be present.…”

“…6,7,12,13,16,17 Regarding absolute values, two separate laboratories reported levels of PEA and OEA in plasma that were excessively high, 18,19 which reached or exceeded the concentrations needed by these ligands to engage PPAR-α as agonists. PEA and OEA are, in fact, considered high-potency ligands of PPAR-α; in heterologous expression systems, these FAEs engage the receptor with median effective concentration (EC 50 ) values of 0.12 μM for OEA and 3 μM for PEA.…”

“…In particular, reported levels of PEA and OEA in serum or plasma of healthy human subjects differ by up to two orders of magnitude, from 5 to 30 pmol per mL 6–17 up to 200 pmol per mL of serum or plasma. 18,19 …”

Lipid Discovery by Combinatorial Screening and Untargeted LC-MS/MS

“…By LC-MS/MS on a triple quadrupole mass spectrometer TSQ Vantage (Thermo Fisher Scientific, San Jose CA) was performed as described22 with minor modifications; MRM transitions and instrument settings are in Supplementary Table S1. NAAsp were quantified by external calibration using unlabeled synthetic compounds.…”

“…To compensate matrix effects standards were spiked into a rat tissue extract in which NAAsp were undetectable. Comparative profiling of NAE by the method of PRM on a hybrid quadrupole – Orbitrap tandem mass spectrometer Q Exactive (Thermo Fisher Scientific) was performed as described22 using inclusion list of precursor m/z (Supplementary Table S1). Rat tissues were obtained from licensed Biomedical Services Facility (MPI of Molecular Cell Biology and Genetics, Dresden).…”