Simultaneous tissue profiling of eicosanoid and endocannabinoid lipid families in a rat model of osteoarthritis

Abstract

We describe a novel LC method for the simultaneous and quantitative profiling of 43 oxylipins including eicosanoids, endocannabinoids, and structurally related bioactive lipids with modified acyl groups. The LC-MS/MS method uses switching at a defined time between negative and positive electrospray ionization modes to achieve optimal detection sensitivity for all the lipids. The validated method is linear over a range of 0.01–5 nmol/g (0.1–50 nmol/g for 2-arachidonoyl glycerol) with intra- and interday precision and accuracy between 1.38 and 26.76% and 85.22 and 114.3%, respectively. The method successfully quantified bioactive lipids in different tissue types in the rat, including spinal cord, dorsal root ganglia (DRGs), knee joint, brain, and plasma. Distinct regional differences in the pattern of lipid measured between tissue types were observed using principle component analysis. The method was applied to analyze tissue samples from an established preclinical rat model of osteoarthritis (OA) pain and showed that levels of 12-hydroxyeicosatetraenoic acid were significantly increased in the OA rat knee joint compared with controls, and that 15-hydroxyeicosatetraenoic acid was significantly increased in the DRGs in the model of OA compared with controls. The developed LC-MS/MS method has the potential to provide detailed pathway profiling in tissues and biofluids where the disruption of bioactive oxylipins may be involved in disease states.

Targeting brain and peripheral plasticity of the lipidome in acute kainic acid-induced epileptic seizures in mice via quantitative mass spectrometry

“…Accordingly, co-extraction and single run quantitative analysis of multiple lipid targets is desirable to circumvent the limitations in sample availability, the pitfalls originating from variability between experimental animal batches and to improve the time and cost-effectiveness of such studies. Recently, a co-extraction method for eCBs and eiCs was developed and applied for rat brain tissues [36].…”

Validation of a tandem mass spectrometry method using combined extraction of 37 oxylipins and 14 endocannabinoid-related compounds including prostamides from biological matrices

“…Furthermore, OxL are more easily ionized in negative mode due to the free carboxylic acid moiety, while eCB and prostamides are more sensitive in positive mode. This constitutes a major drawback of one single LC-MS/MS run, since it requires switching ionization mode (negative/positive) leading to sensitivity loss [8]. However, it was successfully used to quantify 42 compounds in the OxL and eCB metabolomes in a rat model of osteoarthritis [8].…”

“…A variety of physiological processes have been associated with the modulation of these levels, for instance inflammation, pain and appetite [5][6][7]. Interrelations between the OxL and eCB metabolomes have recently attracted attention, not only with regard to their involvement in common physiological processes, but also in terms of their participation in similar enzymatic pathways [8]. Fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) are the main catabolic enzymes for eCB, but studies have revealed a key role of COX-2 activity (especially if FAAH is inhibited) in the presence of anandamide to generate prostamides (prostaglandin ethanolamides) [9,10].…”

“…The following native and deuterated standards were 8,11,14,9(10)EpOME, 12(13)-EpOME, 9(10)-DiHOME, 12(13)-DiHOME, 5 4 and TXB 2 -d 4 . 9,10,13-TriHOME and 9,12,13-TriHOME were obtained from Larodan (Sweden, Malmö).…”

“…Final stock solution concentrations of native standards were: 250 g/mL (2-AG, 2-LG, POEA and LEA), 125 g/mL (OEA, PEA, AEA, DHEA, NAGly, DEA and EPEA), 83 g/mL (SEA), 1 g/mL (PGF 2␣ -EA and PGE 2 -EA) and 10 g/mL (all OxL native standards). Stock solutions of internal standards (IS) were prepared to a final concentration of 40 g/mL (2-AG-d 8 Stock solutions of eCB-related compounds were prepared fresh on a monthly basis and stored at −80 • C, while the same was done for OxL every 6 months. The stock solutions were further diluted in methanol at 10 different calibration levels (Tables S1 and S2, Supporting Information) and prepared fresh before analysis.…”