Electron spin resonance spectroscopic demonstration of the generation of reactive oxygen species by diseased human synovial tissue following ex vivo hypoxia-reoxygenation.

Abstract

Objective-To apply an electron spin resonance (ESR) spectroscopic technique as a means of determinKing the oxidising capacity of reactive oxygen species produced during hypoxia and reoxygenation of diseased human synovial tissue. Methods-Twenty four specimens of fresh synovial tissue were obtained from patients undergoing primary total knee joint replacement and graded according to the degree of inflammation present.Tissue samples were subjected to an ex vivo hypoxia-reoxygenation cycle in the presence of the nitroso based spin trap, 3,5-dibromo-4-nitrosobenzene sulphonate. The degree of oxidation of the spin trap to a stable free radical was determined and folilowed with time. Control samples were subjected to hypoxia only. Results-The results indicate that the oxidising capacity of reactive oxygen species produced by human synovial tissue varies with the degree of inflammation present. Only the more inflamed specimens, from both rheumatoid arthritis and osteoarthritis patients, demonstrated increased production of reactive oxygen species when subjected to a hypoxia-reoxygenation cycle. This change was reduced by both Mason'6 have similarly assigned the three line spectrum generated in such experiments to an oxidation product of DBNBS. In accordance with this assignment, the ESR signal is abolished by reduction with ascorbic acid. '7 In the present study, immunohistochemical analysis was also performed to examine potential relationships between the clinical scoring system used (based on thickness and colour of the synovium), histological features, and the oxidising capacity of reactive oxygen species produced by the synovial tissue specimens following hypoxia-reoxygenation.In view of the potential critical importance of the enzyme xanthine oxidase/dehydrogenase to the process of hypoxic-reperfusion and its localisation to the synovial endothelium,8 we chose to stain our sections in order to identify the vascular endothelium and allow an assessment of vessel density.In addition, using this method we have assessed the capacity of oxypurinol (a competitive inhibitor of xanthine oxidase)'8 and amflutizole (a non-competitive inhibitor),'9 to limit the generation of synovial tissue derived oxidising species following ex vivo hypoxia-reoxygenation.Patients and methods This study was approved by the London Hospital Medical College Ethical Committee.Patients from whom samples were obtained were all undergoing primary total knee joint replacements at the Royal London Hospital. Of the 24 patients studied (age range 48-82), 15 had primary osteoarthritis and nine had rheumatoid arthritis, as diagnosed by the referring physician. Samples of tissue were obtained from the suprapatellar and fat pad regions of the knee joint at surgery. The tourniquet used for the prevention of operative blood loss had been applied for an average of 11 (SD 6) minutes before sample collection. Time from sampling to commencement of the experimental protocol was approximately 30 minutes. The tissue samples were graded clinically as bein...

Free radicals and pathology: current concepts

“…In support of this, we have previously shown that ex vivo simulated hypoxia/reperfusion results in the generation of ESR-detectable ROS, which were inhibited by XO inhibitors [9]. We and others (see Evans and Stefanovic-Racic, this volume) have also reported enhanced nitrite levels in rheumatoid synovial fluid [10], indicating the availability of substrates for NO' generation via XO under hypoxia.…”

Evidence supporting an increased presence of reactive oxygen species in the diseased equine joint

“…Rowley et al (1984) found lipid peroxidation products in the synovial fluid of individuals with rheumatoid arthritis. Singh et al (1995) found that individuals with severe osteoarthritis had increased levels of free radicals produced within the joint during exercise. Auer et al (1993) induced arthritis in horses by carrageenin injection and found that saline-injected joints, which had less of an inflammatory reaction, had fewer lipid peroxidation products present.…”

Somatic mutations in the p53 tumor suppressor gene in rheumatoid arthritis synovium

“…More recently, it has been shown that normal skin can harbor clones of cells carrying p53 mutations (33). Similarly, the RA synovium is chronically exposed to genotoxic insults, such as NO and oxygen radicals that can produce transition mutations by oxidative deamination (34)(35)(36).…”