Evidence Of Acrolein As An Inflammatory Biomarker In Synovial Fluid Of Dogs With Osteoarthritis

Purpose: Endogenous acrolein is considered a toxic aldehyde that is a byproduct of tissue trauma. It has been shown to be present in diseases associated with brain and spinal cord and to exacerbate tissue inflammation and damage in humans and rodents. Anti-acrolein treatments have resulted in clinical improvements by reducing oxidative stress in damaged tissues. The first aim of this pilot study was to evaluate and compare presence of acrolein in the synovial fluid (SF) of dogs with osteoarthritis (OA) and Control joints. The second aim was to evaluate whether acrolein can be used as a potential inflammatory biomarker of OA to discriminate between OA and Control dogs’ SF samples. The final aim was to explore whether use of samples that had been stored in the frozen state for a short period of time versus a long period of time would impact the Results of biomarker comparisons. Methods: Dogs in the OA group were recruited based on presence of radiographic and surgically confirmed non-traumatic secondary OA in a synovial joint; the OA was graded based on radiographs. Dogs in the Control group were recruited based on lack of abnormal findings during complete physical, neurologic and orthopedic examination. Synovial fluid (SF) samples were aseptically aspirated from the joints with secondary OA (OA group) as well as the knees of dogs with healthy joints (Control group); only one joint from each dog was sampled. Samples were frozen immediately in -80°C prior to batch analysis. Due to its short half-life, acrolein detection was performed using a byproduct of its interaction with endogenous lysine in the SF (i.e., FDP-Lysine). Matrix metalloproteinase-2 (MMP-2) was measured as a positive control of inflammation and anti-∝ -1 antitrypsin (A1AT) was measured and used as a loading internal control. All three molecules were measured using the Western blot imaging methodology and normalized to A1AT. Statistical analysis was performed using paired t-test to evaluate differences in biomarkers between OA and Control groups and Spearman test was used to evaluate correlations between radiographic grades in OA group and the measured biomarkers. Results: A total of 18 and 9 SF samples were obtained from the OA group and Control group dogs, respectively. Causes of secondary OA included elbow dysplasia, osteochondrosis dissecans, degenerative cranial cruciate ligament rupture, and patella luxation. The sources of samples included elbow, knee and shoulder joints. Of these samples, four of the OA group and five of the Control group samples had been stored in the frozen state for 4-5 years with the remainder having been in a frozen state for less than 6 months (Table 1). The dogs in the OA group had significantly higher body weight (P = 0.003) and included higher number of neutered males and females (P < 0.001) compared to the Control group dogs. The measured band signal for FDP-lysine and MMP-2 were both significantly higher in the OA group compared to Controls (P = 0.026 and P = 0.048, respectively). Mean+ standard deviation (SD) for FDP-Lysine for the OA group and Control group samples were 0.70(0.23) and 0.52(0.15), respectively. Mean+ SD for MMP-2 for the OA group and Control group samples were 1.17 (0.38) and 0.83(0.26), respectively. The means of measured biomarkers (i.e., FDP-Lysine and MMP-2) in the long-term frozen samples were consistently less than those of recently frozen samples in both groups. However, these differences with regards to duration of the frozen state of samples were not statistically significant (P > 0.05). There was no statistically significant correlation between the radiographic grade of OA and the measured FDP-lysine or MMP-2 biomarkers. Conclusions: This is the first study documenting evidence of acrolein, based on the surrogate biomarker FDP-Lysine, in canine SF samples. The long-term versus short-term storage of SF samples did not impact the ability to detect the measured biomarkers and allowed discrimination between OA and Control groups. The preservation of these biomarkers provided evidence that biobanking samples did not result in significant molecular degradation. The higher level of acrolein in tandem with elevated MMP-2 in the OA group was representative of the inflammatory state of the joint fluid environment compared to Controls. Identification of higher levels of acrolein in the OA group in this pilot study is the first step in evaluating its role as an inflammatory biomarker that may be utilized in OA research as a therapeutic target. Future studies including evaluation of acrolein levels specific to each joint pathology in larger group sizes is warranted.Table 1Source and age of synovial fluid samples for osteoarthritis (OA) and Control groupsGroupJointCause of secondary OADuration of frozen stateShort-termaLong-termbOA (n=18)ElbowElbow dysplasia5-StifledCrCLR(n=11),MPL(n=1)84ShoulderOCD1-Control (n=9)Stifle-45aSamples frozen in -80°C for less than 6 months since collection. b Samples frozen in-80°C for 4-5 years since collection. Elbow dysplasia: fragmented medial coronoid process, osteochondritis dissecans of humeral condyle, ununited anconeus process; dCrCLR: degenerative cranial cruciate ligament rupture; MPL: medial patella luxation; OCD: osteochondritis dissecans of humeral head. Open table in a new tab aSamples frozen in -80°C for less than 6 months since collection. b Samples frozen in-80°C for 4-5 years since collection. Elbow dysplasia: fragmented medial coronoid process, osteochondritis dissecans of humeral condyle, ununited anconeus process; dCrCLR: degenerative cranial cruciate ligament rupture; MPL: medial patella luxation; OCD: osteochondritis dissecans of humeral head.