Profiling the Oxylipin and Endocannabinoid Metabolome by UPLC-ESI-MS/MS in Human Plasma to Monitor Postprandial Inflammation

Abstract

Bioactive lipids, including oxylipins, endocannabinoids, and related compounds may function as specific biochemical markers of certain aspects of inflammation. However, the postprandial responsiveness of these compounds is largely unknown; therefore, changes in the circulating oxylipin and endocannabinoid metabolome in response to a challenge meal were investigated at six occasions in a subject who freely modified her usual diet. The dietary change, and especially the challenge meal itself, represented a modification of precursor fatty acid status, with expectedly subtle effects on bioactive lipid levels. To detect even the slightest alteration, highly sensitive ultra-performance liquid chromatography (UPLC) coupled to electrospray ionization (ESI) tandem mass spectrometry (MS/MS) methods for bioactive lipid profiling was employed. A previously validated UPLC-ESI-MS/MS method for profiling the endocannabinoid metabolome was used, while validation of an UPLC-ESI-MS/MS method for oxylipin analysis was performed with acceptable outcomes for a majority of the parameters according to the US Food and Drug Administration guidelines for linearity (0.9938 < R2 < 0.9996), limit of detection (0.0005–2.1 pg on column), limit of quantification (0.0005–4.2 pg on column), inter- and intraday accuracy (85–115%) and precision (< 5%), recovery (40–109%) and stability (40–105%). Forty-seven of fifty-two bioactive lipids were detected in plasma samples at fasting and in the postprandial state (0.5, 1, and 3 hours after the meal). Multivariate analysis showed a significant shift of bioactive lipid profiles in the postprandial state due to inclusion of dairy products in the diet, which was in line with univariate analysis revealing seven compounds (NAGly, 9-HODE, 13-oxo-ODE, 9(10)-EpOME, 12(13)-EpOME, 20-HETE, and 11,12-DHET) that were significantly different between background diets in the postprandial state (but not at fasting). The only change in baseline levels at fasting was displayed by TXB2. Furthermore, postprandial responsiveness was detected for seven compounds (POEA, SEA, 9(10)-DiHOME, 12(13)-DiHOME, 13-oxo-ODE, 9-HODE, and 13-HODE). Hence, the data confirm that the UPLC-ESI-MS/MS method performance was sufficient to detect i) a shift, in the current case most notably in the postprandial bioactive lipid metabolome, caused by changes in diet and ii) responsiveness to a challenge meal for a subset of the oxylipin and endocannabinoid metabolome. To summarize, we have shown proof-of-concept of our UPLC-ESI-MS/MS bioactive lipid protocols for the purpose of monitoring subtle shifts, and thereby useful to address lipid-mediated postprandial inflammation.

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

“…Our previous methods for profiling the OxL [24] and eCB [25] metabolome separately provided a starting point for further advancement to include a broader range of metabolites, e.g. prostamides, and to increase the sensitivity, with the goal of obtaining a more clinically relevant coverage of the bioactive lipids using smaller sample volumes.…”

“…SPE protocols previously reported for separate extraction of compounds in the eCB [25] and OxL [1,15,24] metabolome were combined and adapted to fit in one single protocol. Briefly, compounds were extracted using Waters Oasis HLB cartridges (60 mg of sorbent, 30 m particle size).…”

“…The conventional accepted range of inter-and intraday accuracy is 80-120% according to US Food and Drug Administration (FDA) guidelines [26]. Accuracy and precision for compounds in the OxL [24] metabolome are given in Table S7 in Supporting Information.…”

“…In a first approach, internal standards for both the OxL and eCB metabolomes were extracted together using the SPE protocol described above. In the second approach, OxL and eCB were extracted separately using our previously reported methods [24,25].…”

“…The OxL separation gradient of the LC method was kept as previously reported [24]. For profiling the eCB metabolome, including the prostamides, the mobile phase was changed from the previous used methanol buffer (10 mM CH 3 COONH 4 ) [25] to the one employed for OxL analysis.…”