Xanthine oxidase response in acute joint injury

Abstract

Purpose: Xanthine oxioreductase (XOR) is a widely distributed molybdoflavoenzyme enzyme that catalyzes the oxidation of hypoxanthine to xanthine and further catalyzes the oxidation of xanthine to uric acid (UA). XOR appears in two distinct interconvertible forms. XOR is constitutively expressed as a dehydrogenase but can be post-translationally modified by reversible thiol oxidation or irreversible proteolytic cleavage to its oxidase form (XO). Circulating XOR exist almost exclusively as the proteolytic generated XO. In the XO form, through the process of oxidizing purines to urate, it becomes a major source of reactive oxygen species (ROS) production. XO generated ROS such as superoxide and H2O2 are well known to cause damage to many cell types, including synoviocytes and chondrocytes. We hypothesized that there would be an increase in synovial fluid (SF) XO activity in acute joint injury with an attendant rise in ROS that could contribute to further joint damage. To our knowledge this is the first study of XO activity in acute joint injury. Methods: Patients: 11 patients without osteoarthritis (OA), (ages 18-29, mean 23) were enrolled in a pilot intervention trial consisting of a single intra-articular knee injection of interleukin-1 receptor antagonist or saline following joint injury with anterior cruciate ligament tear. SF and serum were collected from 9 of these subjects at presentation to the clinic after acute knee injury (mean 15 +/- 7 days) and at the follow-up visit for reconstructive surgery (mean 48 +/- 12 days) for a total of 18 samples. SF from 23 subjects with minimal knee OA (KL grade 0-1, ages 38-81, mean 64) was used as a non injury control group. SF analysis: XO activity and protein carbonyl were measured using kits from Cayman Chemical (Ann Arbor, MI). UA was analyzed using HPLC. As indicators of collagen synthesis and degradation, procollagen II C-propeptide (CPII) and collagen type II telopeptide (CTxII) were measured by ELISA (Ibex, Montreal, Quebec, and IDS, Scottsdale, AZ respectively). Statistical Analysis: Graphpad Prism software (San Diego, CA). Results: The 18 samples from the 9 subjects in the acute injury cohort were treated as a single group for the purpose of this analysis because no effect was observed on any of the above analytes due to treatment group or differences in time course, nor were any trends seen between the two time points. SF XO activity was significantly higher (14X) than serum XO activity in the acute injury group and was significantly higher (5X) than control group SF XO activity (Figure 1). SF carbonyl concentration was significantly higher (2.5X) in the acute injury group than the control group (Figure 2a). Importantly, this indicator of ROS production showed a strong positive association with XO activity in the injury group (Figure 2b). SF UA was significantly lower (1.5X) in the acute injury group than the control group and there was a strong negative correlation of UA with XO activity and carbonyl formation (Figure 3). SF CPII also showed a negative correlation with XO activity while no association was found between SF XO and SF CTxII (Figure 4). Conclusions: This study shows that SF XO activity is increased in acute joint injury and that this increase is strongly associated with an increase in the production of ROS as measured by protein carbonyl content. These data confirm that oxidative stress participates in the pathophysiology of joint damage following acute joint injury. While an increase in UA production might also have been expected, in fact, we observed a negative correlation between XO and UA indicating that any extra UA being produced is being consumed as an antioxidant. This is not surprising, since UA is known to be a potent antioxidant. This increased XO activity may also be contributing to a decrease in type II collagen production thus hindering the repair response of the acutely injured joint. View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)