using high-resolution direct infusion electrospray ionization using fourier transform ion- cyclotron mass spectrometry (DI-ESI-FTICRMS) (Solarix 15T, Bruker) combined with in-depth data-dependent liquid chromatography tandem mass spectrometry (LC-MS/ MS) and LC-MS3 utilizing ion trap MS (IT-MS) (AmaZon speed, Bruker Daltonics). A master list containing almost 1300 masses detected in at least one sample was generated from the FTICRMS data. Multivariate statistical analysis (principal component analysis, PCA) did not show differences based on diagnosis, but nonetheless grouped samples in two separate groups. A strong partial least squares discriminant analysis (PLS-DA) model of the LC-MS data using this ‘natural’ clustering as a classifier revealed several features with an m/z over 800 as important in driving the classification, and these results were confirmed using PLS-DA and orthogonal PLS-DA models of the FTICRMS data. Based on the accurate masses in the FTICRMS data these features were hypothesized to be esterified oxylipids. LC-MS3 analysis of the intact lipids and LC-MS/MS of their hydrolysis products were used to confirm and identify structural isomers. Using this approach five prominent features driving the classification were fully characterized and the presence of diacylphosphatidylcholine containing hydroxylated docosapentaenoic acid PC(16:0/22:5(OH)) was confirmed in human SF.
In Chapter 3 a facile sample preparation protocol and LC-MS/MS platform for the analysis of oxylipids is described. Thirty-three oxylipids, including SPMs, their pathway markers, and pro-inflammatory lipid mediators (LMs) are analyzed within an 11 min single reaction monitoring (SRM) LC-MS/MS run (QTRAP® 6500, Sciex) after a straightforward protein precipitation and dilution. The sample preparation and analysis can be carried out in 96-well format, ensuring high throughput. The validation of the protocol was done using human EDTA plasma, including repeatability, recovery, linearity, matrix effects and accuracy. The platform was subsequently applied in the analysis of SF post-mortem samples, cellular extracts from activated polymorphonuclear (PMN) cells, and whole blood activated in the presence of eicosapentaenoic acid (EPA). The results show that both SPMs and their pathway markers are absent (at least below the lower limit
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of quantification) in post-mortem SF samples from donors not having an arthritic history.The results also show production of pro-inflammatory mediators by PMN upon activation and the possible formation of the pro-resolving mediator resolvin E2 (RvE2) from the in vitro incubation of whole blood with EPA.
As not all the isomers analyzed with the method in Chapter 3 are baseline separated with the applied gradient and C18 column chemistry, and isomers and stereoisomers in particular (diastereomers and enantiomers) are difficult or impossible to resolve using tandem mass spectrometry, we describe an additional separation method in Chapter 4. This platform utilized micro liquid chromatography differential mobility spectrometry tandem mass spectrometry (µLC-DMS-MS/MS) (SelexION™ QTRAP® 5500, Sciex). The DMS adds another separation dimension, thus aiding in the analysis of these oxylipids. Initial DMS optimization on standard material was done using DI and then adjusted to µLC conditions. The DI approach was used to separate two isomeric protectins, PD1 and PDX (10S,17S- diHDHA), and the µLC-DMS-MS/MS method to distinguish between leukotriene B4 (LTB4) and 5S,12S-diHETE in murine peritoneal exudate cell samples, showing that LTB4 is present only after zymosan A injection while its isomer 5S,12S-diHETE is produced after saline (PBS) administration.
In Chapter 5 we discuss different sample handling and storage conditions prior to LC- MS analysis and the effect on measured levels of oxylipids and FAs. Within clinical routines, blood derived samples are widely used for diagnostic and research purposes. In order to guarantee sample integrity and study outcomes it is therefore important to have standardized and validated procedures for blood collection, plasma preparation and its storage. Levels of oxylipids and FA, in platelet-poor plasma aliquots (stored under different conditions) were compared in a longitudinal study, either for short-term storage conditions or long-term. Our data clearly shows a difference between anticoagulants used in plasma preparation, some that could only be explained as an effect of heparin to activate residual platelets in the platelet-poor plasma, not seen in EDTA plasma. Care must also be taken when storing plasma samples for lipidomic analysis, storage at very low temperature (-80 °C) and the addition of methanol directly after sampling are the most important measures to avoid ex vivo synthesis of oxylipids. Finally, the storage-induced oxylipids were analyzed with chiral analysis and residual enzymatic activity found to be one of the possible contributors to the ex vivo formation of oxylipids, even at -20 °C.
Moreover, contrary to the common view of polyunsaturated fatty acid (PUFA) degradation over time due to peroxidation, PUFA levels increased in aliquots over time, instead of decreasing. Addition of methanol prior to storage seemed to have some effect towards minimizing this increase, and therefore we can speculate about lipase activity. This however, as other possible enzymatic activity, warrants further investigation. Finally, Chapter 6 describes a targeted approach to investigate pathways of resolution in knee OA patients, using SF, SF cells (SFCs), and synovial tissue. The inflammation present
in the OA knee joint was characterized by identifying inflammatory cells, cytokines, chemokines, PUFAs and oxylipids present in SF and comparing them to SF from RA patients (control). The data indicates that inflammation is qualitatively and quantitatively different in OA compared to RA, being characterized by a lower inflammatory load (SFCs and cytokines).
Thirty-seven lipids were detected in the soluble fraction of SF, including polyunsaturated fatty acids (PUFAs) and their pro-inflammatory and pro-resolving lipoxygenase (LOX) and cyclooxygenase (COX) pathway markers in both OA and RA patients. Intriguingly, although SPM precursors were readily detectable in the soluble fraction of the SF, no SPM could be detected. In contrast, in the insoluble fraction (cells and undigested matrix) resolvin D2 (RvD2) was detected.
Despite the lower inflammatory load in OA, the efficiency of the generation of (pro-)inflammatory LMs via the COX pathway seemed similar in both patient groups. SFCs of OA patients also contained very low/undetectable amounts of PUFAs and their derivatives when measured directly. Together this suggests that SF inflammatory cells do not significantly add to the levels of these COX-derived LMs in either disease. In contrast, synovial cells (synoviocytes) contained detectable levels even in the absence of extra stimulation, indicating them as a possible source of arachidonic acid (AA)- derived oxylipids in the joint. Oxylipids in SF that were different between OA and RA are generated via 5-LOX or 15-LOX pathways, suggesting a lower activity of these enzymes in OA compared to RA. This is also supported by differences in the ratio between 5- or 15-LOX products and their PUFA precursors. Interestingly, synoviocytes and SFCs could produce 5-LOX and 15-LOX metabolites, indicating these cells as possible source of LM in SF.
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Nederlandse samenvatting
De focus van dit proefschrift ligt op de analyse van (oxy)lipiden. In Hoofdstuk 1 wordt een inleidend overzicht gegeven van lipiden en lipidomics en de daaropvolgende hoofdstukken zijn gericht op de ontwikkeling van lipodomics methoden voor de analyse van geoxideerde lipiden. Deze methoden zijn daarna toegepast, voornamelijk op humaan plasma en synoviaal vocht (SV) monsters. Speciale aandacht ging uit naar de doelgerichte analyse van gehydroxyleerde vetzuren, ontstekingsremmende mediatoren, en andere oxylipiden die aanwezig zijn in inflammatie en zijn resolutie.
Hoofdstuk 2 beschrijft en bespreekt een platformonafhankelijke aanpak voor de analyse