Sandra Gouveia-Figueira

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

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

There is a clinical need for more relevant coverage of bioactive lipids using smaller sample volumes. Therefore, we have validated a tandem mass spectrometry method for combined solid phase extraction of 37 compounds in the oxylipin (OxL) and 14 in the endocannabinoid (eCB) metabolome, as well as prostamides. The limits of quantification (LOQ) for compounds in the eCB metabolome were in the range 0.5-1000 fg on column, intraday accuracy and precision ranges (%) were 83-125 and 0.3-17, respectively, and interday accuracy and precision ranges (%) were 80-119 and 1.2-20, respectively, dependent upon the compound and the concentration studied. Corresponding values for OxL were 0.5 fg-4.2 pg on column (LOQ), 85-115% (inter- and intraday accuracy) and <5% (precision). The combined extraction method was successfully applied to tissues, cell extracts, human plasma and milk samples. A deeper study of levels in elk, pig and cow brain, as well as cow heart and liver revealed tissue and species-specific elevation of eicosanoids: arachidonate diols, 20-HETE and 12(S)-HEPE (cow liver), LTB4 (cow brain), and monohydroxy metabolites (HETEs), epoxides and 5-oxo-ETE in elk brain, which might be caused by factors of stress and/or post-mortem reactions in the tissues.

Serum levels of oxylipins in achilles tendinopathy: an exploratory study.

Background Linoleic acid-derived oxidation products are found in experimental pain models. However, little is known about the levels of such oxylipins in human pain. In consequence, in the present study, we have undertaken a lipidomic profiling of oxylipins in blood serum from patients with Achilles tendinopathy and controls. Methodology/Principal findings A total of 34 oxylipins were analysed in the serum samples. At a significance level of P<0.00147 (<0.05/34), two linoleic acid-derived oxylipins, 13-hydroxy-10E,12Z-octadecadienoic (13-HODE) and 12(13)-dihydroxy-9Z-octadecenoic acid (12,13-DiHOME) were present at significantly higher levels in the Achilles tendinopathy samples. This difference remained significant when the dataset was controlled for age, gender and body-mass index. In contrast, 0/21 of the arachidonic acid- and 0/4 of the dihomo-γ-linolenic acid, eicosapentaenoic acid or docosahenaenoic acid-derived oxylipins were higher in the patient samples at this level of significance. The area under the Receiver-Operator Characteristic (ROC) curve for 12,13-DiHOME was 0.91 (P<0.0001). Levels of four N-acylethanolamines were also analysed and found not to be significantly different between the controls and the patients at the level of P<0.0125 (<0.05/4). Conclusions/Significance It is concluded from this exploratory study that abnormal levels of linoleic acid-derived oxylipins are seen in blood serum from patients with Achilles tendinopathy. Given the ability of two of these, 9- and 13-HODE to activate transient receptor potential vanilloid 1, it is possible that these changes may contribute to the symptoms seen in Achilles tendinopathy.

Multi-platform metabolomics assays for human lung lavage fluids in an air pollution exposure study

AbstractMetabolomics protocols are used to comprehensively characterize the metabolite content of biological samples by exploiting cutting-edge analytical platforms, such as gas chromatography (GC) or liquid chromatography (LC) coupled to mass spectrometry (MS) assays, as well as nuclear magnetic resonance (NMR) assays. We have developed novel sample preparation procedures combined with GC-MS, LC-MS, and NMR metabolomics profiling for analyzing bronchial wash (BW) and bronchoalveolar lavage (BAL) fluid from 15 healthy volunteers following exposure to biodiesel exhaust and filtered air. Our aim was to investigate the responsiveness of metabolite profiles in the human lung to air pollution exposure derived from combustion of biofuels, such as rapeseed methyl ester biodiesel, which are increasingly being promoted as alternatives to conventional fossil fuels. Our multi-platform approach enabled us to detect the greatest number of unique metabolites yet reported in BW and BAL fluid (82 in total). All of the metabolomics assays indicated that the metabolite profiles of the BW and BAL fluids differed appreciably, with 46 metabolites showing significantly different levels in the corresponding lung compartments. Furthermore, the GC-MS assay revealed an effect of biodiesel exhaust exposure on the levels of 1-monostearylglycerol, sucrose, inosine, nonanoic acid, and ethanolamine (in BAL) and pentadecanoic acid (in BW), whereas the LC-MS assay indicated a shift in the levels of niacinamide (in BAL). The NMR assay only identified lactic acid (in BW) as being responsive to biodiesel exhaust exposure. Our findings demonstrate that the proposed multi-platform approach is useful for wide metabolomics screening of BW and BAL fluids and can facilitate elucidation of metabolites responsive to biodiesel exhaust exposure. Graphical AbstractGraphical abstract illustrating the study workflow. NMR Nuclear Magnetic Resonance, LC-TOFMS Liquid chromatography-Time Of Flight Mass Spectrometry, GC Gas Chromatography-Mass spectrometry

Postprandial metabolomics: A pilot mass spectrometry and NMR study of the human plasma metabolome in response to a challenge meal

The study of postprandial metabolism is relevant for understanding metabolic diseases and characterizing personal responses to diet. We combined three analytical platforms - gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) - to validate a multi-platform approach for characterizing individual variation in the postprandial state. We analyzed the postprandial plasma metabolome by introducing, at three occasions, meal challenges on a usual diet, and 1.5 years later, on a modified background diet. The postprandial response was stable over time and largely independent of the background diet as revealed by all three analytical platforms. Coverage of the metabolome between NMR and GC-MS included more polar metabolites detectable only by NMR and more hydrophobic compounds detected by GC-MS. The variability across three separate testing occasions among the identified metabolites was in the range of 1.1-86% for GC-MS and 0.9-42% for NMR in the fasting state at baseline. For the LC-MS analysis, the coefficients of variation of the detected compounds in the fasting state at baseline were in the range of 2-97% for the positive and 4-69% for the negative mode. Multivariate analysis (MVA) of metabolites detected with GC-MS revealed that for both background diets, levels of postprandial amino acids and sugars increased whereas those of fatty acids decreased at 0.5 h after the meal was consumed, reflecting the expected response to the challenge meal. MVA of NMR data revealed increasing postprandial levels of amino acids and other organic acids together with decreasing levels of acetoacetate and 3-hydroxybutanoic acid, also independent of the background diet. Together these data show that the postprandial response to the same challenge meal was stable even though it was tested 1.5 years apart, and that it was largely independent of background diet. This work demonstrates the efficacy of a multi-platform metabolomics approach followed by multivariate and univariate data analysis for a broad-scale screen of the individual metabolome, particularly for studies using repeated measures to determine dietary response phenotype.

Characterisation of (R)-2-(2-Fluorobiphenyl-4-yl)-N-(3-Methylpyridin-2-yl)Propanamide as a Dual Fatty Acid Amide Hydrolase: Cyclooxygenase Inhibitor

Background Increased endocannabinoid tonus by dual-action fatty acid amide hydrolase (FAAH) and substrate selective cyclooxygenase (COX-2) inhibitors is a promising approach for pain-relief. One such compound with this profile is 2-(2-fluorobiphenyl-4-yl)-N-(3-methylpyridin-2-yl)propanamide (Flu-AM1). These activities are shown by Flu-AM1 racemate, but it is not known whether its two single enantiomers behave differently, as is the case towards COX-2 for the parent flurbiprofen enantiomers. Further, the effects of the compound upon COX-2-derived lipids in intact cells are not known. Methodology/Principal Findings COX inhibition was determined using an oxygraphic method with arachidonic acid and 2-arachidonoylglycerol (2-AG) as substrates. FAAH was assayed in mouse brain homogenates using anandamide (AEA) as substrate. Lipidomic analysis was conducted in unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Both enantiomers inhibited COX-2 in a substrate-selective and time-dependent manner, with IC50 values in the absence of a preincubation phase of: (R)-Flu-AM1, COX-1 (arachidonic acid) 6 μM; COX-2 (arachidonic acid) 20 μM; COX-2 (2-AG) 1 μM; (S)-Flu-AM1, COX-1 (arachidonic acid) 3 μM; COX-2 (arachidonic acid) 10 μM; COX-2 (2-AG) 0.7 μM. The compounds showed no enantiomeric selectivity in their FAAH inhibitory properties. (R)-Flu-AM1 (10 μM) greatly inhibited the production of prostaglandin D2 and E2 in both unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Levels of 2-AG were not affected either by (R)-Flu-AM1 or by 10 μM flurbiprofen, either alone or in combination with the FAAH inhibitor URB597 (1 μM). Conclusions/Significance Both enantiomers of Flu-AM1 are more potent inhibitors of 2-AG compared to arachidonic acid oxygenation by COX-2. Inhibition of COX in lipopolysaccharide + interferon γ- stimulated RAW 264.7 cells is insufficient to affect 2-AG levels despite the large induction of COX-2 produced by this treatment.

Association between plasma concentrations of linoleic acid-derived oxylipins and the perceived pain scores in an exploratory study in women with chronic neck pain

BackgroundChronic musculoskeletal pain may be associated with changes in the balance of algogenic and anti-nociceptive compounds, and such changes may be visible in plasma samples. We have undertaken an exploratory study to measure the levels of endocannabinoids, related N-acylethanolamines and oxylipins (primarily those derived from linoleic acid) in plasma samples from women with chronic neck pain (NP) and chronic widespread pain (CWP), and to investigate whether the observed levels are associated with the pain experienced by these women.MethodsBlood samples from 35 women with NP, 15 with CWP and 27 age-matched controls were analysed for the lipids using ultra performance liquid chromatography coupled to tandem mass spectrometry. Current pain (“NRSday”) and the average pain during the last week (“NRSweek”) were rated by the participants using a numerical rating scale.ResultsThere were no significant differences in the plasma concentrations of the fifteen lipids investigated between the women with pain and the controls. However, significant correlations were seen for the NP group between the NRSday scores and the plasma concentrations of the linoleic acid derivatives 9- and 13-hydroxy-octadecadienoic acid (Spearman’s rho values 0.51 [P = 0.0016]) and 0.53 [P = 0.0011], respectively).ConclusionsThe data obtained in this exploratory study indicate that although no group differences are seen in plasma lipid concentrations, there is an association between the NRSday scores and the 9- and 13-hydroxy-octadecadienoic acid levels. Whether or not the association reflects a causality (i.e. that the circulating lipids contribute to the perceived pain of the pain participants), requires further investigation.

The influence of monoacylglycerol lipase inhibition upon the expression of epidermal growth factor receptor in human PC-3 prostate cancer cells.

BackgroundIt has been reported that direct activation of the cannabinoid CB1 receptor in epidermal growth factor (EGR)-stimulated PC-3 prostate cancer cells results in an anti-proliferative effect accompanied by a down-regulation of EGF receptors (EGFR). In the present study, we investigated whether similar effects are seen following inhibition of the endocannabinoid hydrolytic enzyme monoacylglycerol lipase (MGL).ResultsCB1 receptor expression levels were found to differ greatly between two experimental series conducted using PC-3 cells. The monoacylglycerol lipase inhibitor JZL184 increased levels of 2-arachidonoylglycerol in the PC-3 cells without producing changes in the levels of anandamide and related N-acylethanolamines. In the first series of experiments, JZL184 produced a small mitogenic effect for cells that had not been treated with EGF, whereas an anti-proliferative effect was seen for EGF-treated cells. An anti-proliferative effect for the EGF-treated cells was also seen with the CB receptor agonist CP55,940. In the second batch of cells, there was an interaction between JZL184 and CB1 receptor expression densities in linear regression analyses with EGFR expression as the dependent variable.ConclusionsInhibition of MGL by JZL184 can affect EGFR expression. However, the use in our hands of PC-3 cells as a model to investigate the therapeutic potential of MGL inhibitors and related compounds is compromised by their variability of CB1 receptor expression.

Oxylipins, endocannabinoids, and related compounds in human milk: Levels and effects of storage conditions.

The presence of fatty acid derived oxylipins, endocannabinoids and related compounds in human milk may be of importance to the infant. Presently, clinically relevant protocols for storing and handling human milk that minimize error and variability in oxylipin and endocannabinoid concentrations are lacking. In this study, we compared the individual and combined effects of the following storage conditions on the stability of these fatty acid metabolites in human milk: state (fresh or frozen), storage temperature (4 °C, -20 °C or -80 °C), and duration (1 day, 1 week or 3 months). Thirteen endocannabinoids and related compounds, as well as 37 oxylipins were analyzed simultaneously by liquid chromatography coupled to tandem mass spectrometry. Twelve endocannabinoids and related compounds (2-111 nM) and 31 oxylipins (1.2 pM-1242 nM) were detected, with highest levels being found for 2-arachidonoylglycerol and 17(R)hydroxydocosahexaenoic acid, respectively. The concentrations of most endocannabinoid-related compounds and oxylipins were dependent on storage condition, and especially storage at 4 °C introduced significant variability. Our findings suggest that human milk samples should be analyzed immediately after, or within one day of collection (if stored at 4 °C). Storage at -80 °C is required for long-term preservation, and storage at -20 °C is acceptable for no more than one week. These findings provide a protocol for investigating the oxylipin and endocannabinoid metabolome in human milk, useful for future milk-related clinical studies.

Plasma levels of the endocannabinoid anandamide, related N-acylethanolamines and linoleic acid-derived oxylipins in patients with migraine

There is evidence that patients with migraine have deficient levels of the endogenous cannabinoid receptor ligand anandamide (AEA). It is not known, however, if this is a localised or generalised phenomenon. In the present study, levels of AEA, related N-acylethanolamines (NAEs) and linoleic acid-derived oxylipins have been measured in the blood of 26 healthy women and 38 women with migraine (26 with aura, 12 without aura) who were matched for age and body-mass index. Blood samples were taken on two occasions: the first sample near the start of the menstrual cycle (when present) and the second approximately fourteen days later. For a subset of migraine patients, two additional blood samples were taken, one during a migraine attack and one approximately 1 month later (to be at the same stage in the menstrual cycle, when present). NAEs and oxylipins were measured by liquid chromatography coupled to mass spectrometry. Twenty-nine lipids were quantified, of which 16 were found to have a high reproducibility of measurement. There were no significant differences in the levels of AEA, the related NAEs stearoylethanolamide and oleoylethanolamide or any of the nine linoleic acid-derived oxylipins measured either between migraine patients with vs. without aura, or between controls and migraine patients (after stratification to take into account whether or not the individuals had regular menstruation cycles) in either of the first two samples. Levels of linoleoylethanolamide were lower in the patients with vs. without aura on the second sample but not in the first sample, but the biological importance of this finding is unclear. Due to time-dependent increases in their concentrations ex vivo prior to centrifugation, AEA and oleoylethanolamide levels in the samples collected during migraine attacks were not analysed, but for the other fourteen lipids, there were no significant differences in plasma concentrations during migraine vs. one month later. It is concluded that migraine is not associated with a generalised (as opposed to localised) deficiency in these lipids.