The online version of this article (doi:10.1186/ar3427) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
JCdG carried out the sample collection and extractions, participated in method optimization, and drafted the manuscript. CHAvdL carried out the HPLC-MS/MS optimization and data analysis and performed the statistical analysis. PRvW participated in the design and coordination of the study and helped to draft the manuscript. All authors read and approved the final manuscript.
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) E2 was significantly lower in NSAID vs. placebo treated samples at all time points; PGE1, 11-hydroxyeicosatetraenoic acid (11-HETE) and 13,14-dihydro-15keto PGF2α were reduced at 8 and 24 hours by NSAID treatment; while 15-HETE, 6-keto PGF1α, PGF2α, 13,14-dihydro-15keto PGE2 and thromboxane B2 (TXB2) were reduced at the 8 hour time point only. An interesting pattern was seen for Leukotriene B4 (LTB4), 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 PGE2 or to the early inflammatory phase.
Additional file 1: Calibration lines - Standard curve equations and correlation coefficients for LC-ESI-MS/MS analysis of eicosanoid standards. Calibration lines for liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis were prepared by diluting stock solutions to final concentrations of 100 pg/μL, 50 pg/μL, 25 pg/μL, 10 pg/μL, 5 pg/μL, 2 pg/μL and 1 pg/μL. The internal standard (IS; 16,16-dimethyl prostaglandin F2α) was prepared in ethanol (2 ng/μL) and added to all composite standards at a final concentration of 100 pg/μL. (PDF 49 KB)13075_2011_3185_MOESM1_ESM.PDF
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- A targeted lipidomics approach to the study of eicosanoid release in synovial joints
Janny C de Grauw
Chris HA van de Lest
Paul René van Weeren
- BioMed Central
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