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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. The study design.

After an overnight fast, blood was collected and thereafter the challenge meal was consumed. Postprandial blood collection was done at 0.5, 1, and 3 h after finished meal. The meal challenge was repeated at six occasions in total. Hour of blood collection varied between experiments, illustrated by time spans.

Fig 2. Comparison of the postprandial response in NAGly (eCB) plasma levels in one subject on usual (vegan) and modified (vegetarian) diet, respectively.

*p

Fig 3. MRM chromatograms of (A) NAGLy in a standard solution (136 pg on column) and (B) a representative plasma sample together with corresponding MS spectra (inserts).

Fig 4. Baseline and postprandial response levels of endocannabinoid significantly different for a subject on usual diet (A), and on modified diet (B).

Values represent the mean ± SEM (n = 3 for each diet and time point. ****p

Fig 5. MRM chromatograms of each analyte analyzed in a standard solution mixture (A) and separation of critical pairs of isomers (B).

Fig 6. Average recovery rates for deuterated internal standards (IS) in PBS (n = 5) and human plasma (n = 24) Expressed as mean±SEM.

Fig 7. Significant different baseline levels of TXB2 (p = 0.001) in plasma collected at usual (n = 3) and modified (n = 3) background diets, respectively.

Values represent the mean ± SEM.

Fig 8. Comparison of the postprandial oxylipin plasma levels in one subject on a usual and modified diet, respectively.

* indicates p

Fig 9. Baseline and postprandial response levels significantly different of oxylipins for a subject on usualdiet (A), and on modified diet (B).

Values represent the mean ± SEM (n = 3 for each diet and time point). Brackets indicates significantly different time points: ****p

Fig 10. Oxylipin and endocannabinoid profiles in the postprandial state recapitulated as orthogonal scores (t[1] and to[1]) calculated by OPLS-DA.

Time after challenge meal (in hours) is found next to each sample. The subject displayed different oxylipin and endocannabinoid metabolomes on usual (grey circles) vs modified (black squares) background diet. Model assessment parameters were: 1 predictive and 1 orthogonal component, p-value calculated by CV-ANOVA: 0.033, total systematic variation among the metabolites captured by the model (R2X): 0.463, total systematic variation between the diets captured by the model (R2Y): 0.815, predictive ability of the model (Q2): 0.53.