Effects of bisphosphonate administration on synovial metabolome of juvenile horses challenged with intra-articular lipopolysaccharide

The objective of this study was to evaluate the synovial metabolome in juvenile exercising horses treated with the bisphosphonate clodronate disodium (OSPHOS®) following an intra-articular lipopolysaccharide (LPS) challenge. Treatment groups consisted of 8 control horses that received saline injections (CON) and 8 horses that received clodronate disodium (CD; 1.8 mg/kg BW, IM) on d 0, 42, 84, and 126 of a 140-d study. Horses underwent a progressive exercise regimen that culminated with an intra-articular LPS challenge on d 126. Radial carpal joints of each horse were injected with 0.8 mL of either 0.5 ng LPS derived from Escherichia coli O55:B5 or sterile lactated Ringer's (LRS) solution as a contralateral control. Synovial fluid was collected before LPS injection (h 0) and 6, 12, 24, and 336 h post injection. Synovial fluid samples were extracted using 1:1 water:acetonitrile liquid-liquid extraction. Sample extracts were analyzed using high-performance liquid chromatography quadrupole time-of-flight (HPLC-qTOF) reverse-phase chromatography in positive ion mode. Significant (P ≤ 0.05) metabolites were compared with a list of standards by retention time, m/z ratios, and molecular features. Of the 83 significant (P ≤ 0.05) compounds, 58 were annotated by Wiley and National Institute of Standards and Technology metabolite libraries. Of the annotated compounds there were 14 lipid and 12 amino acid metabolites, with the remainder of unknown origin. Total metabolites of the CD and LPS treatments were segregated by partial least squares discriminate analysis. The principal component analysis discriminant analysis segregated CD and CON samples along component 1, which accounted for 89.64% of the total variation where most metabolites were upregulated in CD compared with CON. Upregulated lipid metabolites included fatty acyls, fatty esters, prenols, and sphingolipids. There was a treatment × time × carpus interaction where 6 h after LPS injection, the relative metabolite abundance was greater in CD vs. CON in the LPS joint (P ≤ 0.05). These metabolites included creatine (P = 0.03), guanosine (P = 0.01), and 2 amino acid metabolites (P = 0.01; P = 0.04). These results indicate that inflammatory progression stimulated by intra-articular LPS increases amino acid and lipid metabolite abundance in the synovial metabolome. Further, CD had increased lipid and amino acid metabolism compared with CON in both the LPS and LRS injected joints. Further investigation of the equine synovial metabolome is warranted in view of this initial observation that bisphosphonate administration upregulated synovial metabolite abundance.