A targeted lipidomics approach to the study of eicosanoid release in synovial joints

Lipid mediators of inflammation play an important role in the local inflammatory response associated with inflammatory arthritides as well as orthopedic arthropathies [1]. Of these mediators, the E-series prostaglandins (most notably PGE₂) are most noted in arthritis research for their pro-inflammatory and pro-nociceptive actions in synovial joints [2, 3].

However, COX and LOX enzyme activity within the arachidonic acid cascade generates a range of eicosanoid mediators that have widely varying biological actions, including anti-inflammatory and pro-resolving effects [4, 5]. In recent years, more light has been shed on the specific actions of individual eicosanoids in arthritis, and several of these (including PGE₂) have emerged as janus-faced mediators with pro-inflammatory or anti-inflammatory effects depending effects, depending on concentration and receptor subtype engagement [6, 7]. Reduction of PGE₂ production is the classical mode of action of anti-inflammatory agents like non-steroidal anti-inflammatory drugs (NSAIDs) that are commonly used in medical management of (osteo)arthritis, and numerous studies have demonstrated a lower PGE₂ concentration in synovial fluid (SF) following NSAID treatment [8‐10]. However, by inhibition of COX activity, these drugs are likely not only to affect PGE₂ production but also to interfere with the production of mediators with differential effects that are generated by the same enzymatic pathways. Indeed, in an early study, NSAID (naproxen) treatment tended to reduce not only PGE₂ but also TXB₂ and 6-keto PGF₁α concentration in the SF of human patients with rheumatoid arthritis [8].

The investigation of the potential involvement of individual eicosanoids in disease states relies on sensitive and specific assays to measure these products in biological fluids. While antibody-based assays for individual eicosanoids are commonly employed, these suffer from cross-reactivity issues and may produce misleading results in complex biological samples [11]. Moreover, they can be used for analysis of only one metabolite at a time, restricting the amount of biological information obtained as SF sample volume tends to be a limiting factor.

In this report, we describe the application of recently developed high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) mediator lipidomics techniques to the study of eicosanoid release in equine synovial joints. To evaluate the relative abundance of these lipid mediator species in normal and inflamed joints and investigate the effects of COX inhibition on eicosanoid profiles, we performed a longitudinal study of SF lipid mediator composition in healthy horses over the course of experimentally induced transient synovitis with and without oral NSAID treatment.

5(S)-hydroxyeicosatetraenoic acid (HETE), 8(S)-HETE, 11(S)-HETE, 12(S)-HETE, and 15(S)-HETE; leukotriene D₄ (LTD₄), LTE₄; LTB₄; 5(S)6(R)15(S)-lipoxin A₄ (LXA₄); prostaglandin E₁ (PGE₁), 6-keto prostaglandin F₁α (6-keto PGF₁α), prostaglandin D₂ (PGD₂), prostaglandin E₂ (PGE₂), prostaglandin F₂α (PGF₂α), 11β-prostaglandin F₂α (11β-PGF₂α), prostaglandin F₂β (PGF₂β), prostaglandin J₂ (PGJ₂), 15-deoxy-Δ12,14-prostaglandin J₂ (15-deoxy-Δ12,14-PGJ₂), thromboxane B₂ (TXB₂), 13,14-dihydro-15-keto PGF₂α, 13,14-dihydro-15-keto PGE₂, 13,14-dihydro-15-keto PGD₂, and 16,16-dimethyl PGF₂α were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). HPLC-grade solvents (acetonitrile and methanol) were from Biosolve (Valkenswaard, The Netherlands), and glacial acetic acid and all other chemicals used for sample extraction and preparation were from Sigma-Aldrich (St. Louis, MO, USA). Solid-phase extraction columns (LiChrolut RP-18; 100-mg column bed) were purchased from Merck (Darmstadt, Germany).

In this report, we describe the application of mediator lipidomics techniques to the study of SF eicosanoid profiles in normal and inflamed equine joints. Furthermore, we illustrate the use of the developed LC-ESI-MS/MS analysis to investigate the effects of NSAID treatment in acute synovitis.

We identified and quantitated 14 individual eicosanoids in SF extracts by using MRM. The MRM transitions used to identify individual compounds in this study were confirmed by literature sources [15‐23]; unfortunately, as previously outlined by Murphy and colleagues [20], many eicosanoids have very similar or even identical (isomeric) chemical structures and therefore a single m/z transition may not be as specific to individual compounds as desired. Future experiments employing information-dependent acquisition in combination with enhanced product ion detection settings could be used to enhance analyte identification [24]. Alternatively, the use of more than one m/z transition per compound (as was done in the present case for TXB₂ and PGE₂) could aid in definitive identification.

The recovery of analytes in this study was estimated by comparing peak areas of the IS in spiked and extracted SF samples with the corresponding standard solution analyzed without extraction. Although this provides an indication of analyte loss over the extraction procedure, it does not account for possible differences in recovery or degradation between individual analytes. While it is certainly not uncommon to use only one IS in studies of multiple analytes [15, 21, 25], it would be preferable to use stable isotope-labeled standards for each analyte under investigation or to use one such labeled standard per class of mediators targeted [24]. However, the 16,16-dimethyl PGF₂α we employed does combine several advantages for use as an IS in the current application: It elutes at an RT close to that of many analytes of interest, it shows good stability at room temperature and at -20°C and -80°C (de Grauw, unpublished observations), and it is not normally found in SF and is not known to have a biological function in synovial joints.

In addition to rapid enzymatic conversion and degradation of lipid mediators in vivo, the reliable analysis of eicosanoids in body fluids may be hampered by ex vivo degradation of labile species [24]. This was recently demonstrated for PGD₂, which was shown to be far more susceptible to chemical decomposition at room temperature and at -20°C than PGE₂ [23]. Hence, relative quantities of these mediators in extracted samples may also reflect selective degradation of one over the other, and absolute levels should be interpreted with caution; the same might be true for other eicosanoids, the stability of which has not been exhaustively addressed. For instance, the current extraction procedure and HPLC solvent system are not optimized for quantitative detection of cysteinyl LTs [25].

We positively identified 14 eicosanoids in SF extracts and found that the concentrations of many of these were significantly elevated in inflamed joints compared with normal (baseline) values (Figures 5 and 6). As LPS induces marked influx of leukocytes (predominantly neutrophils [9]) into the joint space, eicosanoid species detected may partly reflect release by infiltrating cells rather than release from articular sources; however, articular cartilage and, especially, synovial fibroblasts are apt producers of a great number of these mediators [17, 26, 27], and therefore having the means to detect these will aid in future studies of spontaneous disease.

SF eicosanoid profiles changed dramatically upon synovitis induction, as illustrated by the LDA plots showing marked separation between samples taken at different time points. Release of individual prostanoids, HETEs, and LTB₄ over the course of transient synovitis did not reveal marked temporal differences between these classes of mediators, although there was a trend toward early response of prostanoids versus a somewhat more protracted response of HETEs and LTB₄. PGE₁ and PGE₂ as well as PGJ₂ (which is an intermediate breakdown product of PGD₂ and which is known to have anti-inflammatory properties [28, 29]) showed a more prolonged elevation over the first 24 hours of synovitis than other prostanoids. Although our method quantitatively detected 15-deoxy-Δ12,14-PGJ₂ in stock standards and in spiked SF, this anti-inflammatory PG was not detected in SF extracts and hence we cannot comment on its temporal release pattern. The same was true for LXA₄, another endogenous anti-inflammatory and pro-resolving mediator [5]. Some of these species may have escaped detection because of rapid enzymatic conversion or chemical instability as noted above; thus, the precise release kinetics of pro- versus anti-inflammatory eicosanoid species in acute synovitis will need to be addressed in future studies that include even earlier sampling time points or alternative extraction steps or both.

The observed differences between NSAID- and placebo-treated samples clearly demonstrate that the effects of COX inhibitors on synovial eicosanoid release are not limited to PGE₂ reduction. Concentrations of many other prostanoids, including PGE₁, PGD₂, PGJ₂, PGF₂α, 6-keto PGF₁α, and TXB₂, were also significantly lower in NSAID- versus placebo-treated SF samples, particularly in the acute phase of synovitis, and this agrees with and extends previous findings in SF of human subjects treated with naproxen [8]. The observed reduction in PGE₂ and 6-keto PGF₁α (the stable main metabolite of prostacyclin) with NSAID treatment is likely to contribute to analgesic efficacy [30]. Inhibition of PGE₂ and PGE₁ production could limit the negative feedback of these two mediators on matrix metalloproteinase (MMP) production by synovial fibroblasts [28]; however, as meloxicam itself inhibits synovial MMP activity [9], this will not have clinical implications. The consequences of TXB₂, PGD₂, and PGJ₂ inhibition are harder to predict because their roles in arthritis have been less well studied.

Our results for LTB₄ and 5-, 12-, and 15-HETE are interesting because these mediators are all products of the LOX pathway but proved to be differentially affected by NSAID treatment. Both LTB₄ and 5-HETE are downstream products of 5-LOX, 12-HETE is produced through 12-LOX action, and 15-HETE is produced by 15-LOX (while both 11- and 15-HETE can also be produced by COX [31]). As seen in Figure 6, 12-HETE concentration did not change at all over the course of transient synovitis, whereas the concentration of 15-HETE was significantly elevated in the acute phase and reduced by NSAID treatment. Interestingly, the concentration of LTB₄ was significantly higher at 8 hours in SF of NSAID-treated versus placebo-treated horses, and 5-HETE showed a similar trend. A transient increase in LTB₄ release has also been found in cultured synovial membrane and cartilage explants treated with certain COX inhibitors [28, 32] and has been suggested to be due to 'shunting' of arachidonic acid away from COX-mediated PG production toward LOX-mediated LT production [28]. However, our findings suggest that this is an oversimplification since such a general shunt would have resulted in elevated concentrations of all LOX-generated mediators rather than some of them. Perhaps more likely, different LOX isoforms or additional enzymes (or both) that are located within the arachidonic acid cascade and that act upon intermediate metabolites (for example, phospholipid hydroperoxide glutathione peroxidase) may be differentially affected by NSAID treatment. Interestingly, LTB₄ elevation was no longer observed at later time points, and, at 168 hours, meloxicam actually reduced LTB₄ concentration. The biological or clinical implications of this transient rise in LTB₄ upon therapeutic COX inhibition remain to be established [28] and are likely to be complex; while LTB₄ plays a crucial role in driving inflammatory arthritis [33], it may also act to rescue COX-generated mediator production important to resolution of inflammation [34].

Altogether, this study represents a first attempt at simultaneous quantification of more than 20 lipid mediators in SF samples. Despite the more complex data analysis and inherent difficulty in identification of multiple closely related analytes in complex biological samples, the ESI-LC-MS/MS method we employed shows obvious advantages over enzyme-linked immunosorbent assay-based techniques that require relatively large volumes of SF for detection of one selected mediator at a time, while unaccounted-for analyte loss during sample extraction and cross-reactivity between analytes may provide misleading results [15, 24]. The current approach allows high-throughput screening of SF samples by using an IS for estimation of extraction efficiency and requires only 300 μL of SF to analyze a much wider spectrum of eicosanoids, thus enabling the detection of mediators and treatment effects that otherwise would have escaped attention.

We report the application of a sensitive HPLC-MS/MS technique for the simultaneous detection of more than 20 eicosanoids in SF. Extraction efficiency was deemed adequate, and the method showed good linearity and sensitivity. The application of this method to SF samples from horses with experimentally induced synovitis treated with NSAID or placebo confirmed local release of many more eicosanoids than PGE₂ alone over the course of transient synovitis and revealed differential effects of NSAID treatment on several of these mediators: PGE₂ was significantly lower in NSAID- versus placebo-treated samples at all time points; PGE₁, 11-HETE, and 13,14-dihydro-15-keto PGF₂α were reduced throughout the acute phase (8 and 24 hours) by NSAID treatment; whereas 15-HETE, 6-keto PGF₁α, PGF₂α, 13,14-dihydro-15-keto PGE₂, and TXB₂ were reduced at the earliest time point only. An interesting pattern was seen for LTB₄, in which NSAID treatment caused an initial increase at 8 hours but a significant reduction at 168 hours.