Determination of cytidine deaminase activity in synovial fluid by HPLC

✍ Scribed by Karl E. Herbert; David L. Scott; David Perrett

• Publisher: Elsevier Science
• Year: 1989
• Tongue: English
• Weight: 568 KB
• Volume: 7
• Category: Article
• ISSN: 0731-7085
• DOI: 10.1016/0731-7085(89)80118-9

✦ Synopsis

A reversed-phase high-performance liquid chromatographic method for the determination of cytidine deaminase activity in synovial fluid is described. Diluted synovial fluid was incubated for 10 min at 56 degrees C with 0.4 mM cytidine. The protein in 5 vol of incubate was then precipitated using 1 vol of trichloroacetic acid (20% m/v) and the substrate, cytidine, and the product, uridine, were determined in the resultant supernatant. These substances were separated by reversed-phase HPLC using 0.05 M potassium dihydrogen orthophosphate (pH 6.5) containing methanol (3% v/v) and were detected at 280 nm. The enzyme activity was determined by measuring uridine formation. The effects of substrate concentration, pH and reaction temperature on uridine formation are described.