Articular tissues are capable of producing a range of eicosanoid mediators, each of which has individual biological effects and may be affected by anti-inflammatory treatment. We set out to develop and evaluate a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) approach for the simultaneous analysis of multiple eicosanoid lipid mediators in equine synovial fluid (SF), and to illustrate its use for investigation of the in vivo effects of non-steroidal anti-inflammatory drug (NSAID) treatment.
Synovial fluid samples were obtained from normal joints of 6 adult horses at baseline (0 hr) and at 8, 24 and 168 hours after experimental induction of transient acute synovitis, with horses treated once daily with oral NSAID (meloxicam, 0.6 mg/kg) or placebo. Following solid-phase extraction, SF lipid mediator quantitation was based on liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis, and results were compared between disease states using linear discriminant analysis (LDA) and analysis of variance (ANOVA) with multiple comparisons corrections.
Of a total of 23 mediators targeted, 14 could be reliably identified and quantified in SF samples based on detection of characteristic fragment ions at retention times similar to those of commercial standards. LDA analysis of baseline, 8, 24 and 168 hour synovial fluid samples revealed a separation of these groups into discrete clusters, reflecting dynamic changes in eicosanoid release over the course of synovitis. Prostaglandin (PG) E(2) was significantly lower in NSAID vs. placebo treated samples at all time points; PGE(1), 11-hydroxyeicosatetraenoic acid (11-HETE) and 13,14-dihydro-15keto PGF(2)α were reduced at 8 and 24 hours by NSAID treatment; while 15-HETE, 6-keto PGF(1)α, PGF(2)α, 13,14-dihydro-15keto PGE(2) and thromboxane B(2) (TXB(2)) were reduced at the 8 hour time point only. An interesting pattern was seen for Leukotriene B(4) (LTB(4)), NSAID treatment causing an initial increase at 8 hours, but a significant reduction by 168 hours.
The described method allows a comprehensive analysis of synovial fluid eicosanoid profiles. Eicosanoid release in inflamed joints as well as differences between NSAID treated and placebo treated individuals are not limited to PGE(2) or to the early inflammatory phase.