Metabolite profiles discriminate between acl injured cases and uninjured controls within the first year following injury and surgery

Abstract

Purpose: Early biological changes at the metabolite level following acute traumatic knee joint injury may influence the initiation and progression of post-traumatic osteoarthritis. The overall objective of this study was to define the metabolite profile in young and physically active military service members following anterior cruciate ligament (ACL) rupture in comparison to uninjured matched controls. Methods: As part of a larger prospective cohort study we collected biospecimen samples (e.g., serum, plasma, etc.) at the time of ACL injury, the time of ACL reconstruction surgery, and at 6 months and 12 months post-surgery in ACL injured cases and at comparable time points in uninjured controls matched for sex, age (±2 years), height (±5.08 cm), and weight (±6.80 kg). We examined plasma samples from the first 30 ACL injured cases and 30 uninjured matched controls that had complete follow-up at all time points. All sample preparation was completed in batches of 20 samples to maintain sample extraction and derivatization stability prior to comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC×GC-TOFMS) analysis. Plasma samples at each time point were analyzed using the GC×GC-TOFMS method to identify important metabolites that might discriminate between ACL injured cases and uninjured matched controls. Fisher-Ratios were calculated as the group-to-group variance of the detected signal divided by the sum of the within-group variance of the signal and compared between groups at each time point to identify potentially important metabolites. Quantitative analysis for Fisher-Ratio selected metabolites was obtained via PARAFAC, a chemometric deconvolution algorithm, to obtain the metabolite peak areas. Testing for statistical significance of the concentration ratios using the peak areas for the key metabolites was based on t-tests with p-values using Microsoft Excel 365 ProPlus. Following metabolite quantification, the top 10 metabolites identified were carried forward and examined using principal component analysis on metabolite peak areas using PLS Toolbox 8.6. Results: Among the 30 ACL injured cases, 18 were males (60%) and the average age, height and weight was 19.35±1.22 years, 175.98±37.36 cm, and 78.93±16.95 kg. Uninjured controls for each case were matched for sex, age (±2 years), height (±5.08 cm), and weight (±6.80 kg). The top 10 metabolites identified via Fisher-Ratio analyses are presented in the table. Eight of the 10 metabolites were significantly different between ACL injured cases and uninjured matched controls at the time of injury, five were different at the time of ACL reconstruction surgery, six were different 6 months post-surgery, and eight were different 12 months-post surgery. L-methionine, Glyoxylic acid, and Malonic acid concentrations were significantly lower across all time points in ACL injured cases compared to uninjured matched controls. L-Lysine concentrations were significantly higher in ACL injured cases across all time points. Succinic acid concentrations were significantly higher in ACL injured cases at the time of injury and at 6 months and 12 months post-surgery. Arachidonic acid concentrations were significantly higher in ACL injured cases at the time of injury, the time of surgery, and 12 months post-surgery. The total percentage of explained variance by the first two principal components was 55.0% at the time of ACL injury, 58.0% at the time of ACL reconstruction surgery, 59.8% at 6 months post-surgery, and 56.6% 12 months post-surgery. As demonstrated in the figure, modest separation was observed between ACL injured cases and uninjured matched controls at the first two time points (time of injury and surgery) and notable separation was observed at the latter two time points (6 months and 12 months post-surgery). The proportion of cumulative explained variance with the first four principal components improved to 78.2% to 84.5% across all time points. Conclusions: Plasma concentrations for several metabolites appear to differentiate between ACL injured cases and uninjured controls matched for sex, age, height, and weight. This differentiation between groups appears to improve at 6 and 12 months following ACL reconstruction. The metabolites identified may provide insight into the pathways associated with the initiation and progression of post-traumatic osteoarthritis following ACL injury; however, further research and longer term follow-up in this cohort is needed to confirm this.View Large Image Figure ViewerDownload Hi-res image Download (PPT)