Optimization of an enzymatic method for the determination of lysosomal N-acetyl-β-D-hexosaminidase and β-glucuronidase in synovial fluid

Background: Our goal was to develop a suitably sensitive assay for N-acetyl-beta-D-hexosaminidase ( HEX) and beta-glucuronidase to allow their use as markers of joint diseases. Methods: We optimized a spectrophotometric method for the determination of lysosomally derived HEX and beta-glucuronidase in synovial fluid on a microplate reader to improve its utility. HEX and beta-glucuronidase act on the 4-nitrophenyl derivatives N-acetyl-beta-glucosamine and beta-D-glucuronide, respectively, to produce 4-nitrophenol, which can be measured at 405 nm on a microplate reader. Results: Maximum enzyme activity was observed at pH 4.7 in a citrate-phosphate buffer for HEX and at pH 4.5 in an acetate buffer for b-glucuronidase. A 10-mu L sample with 30 mu L of substrate solution and 40 mL of appropriate buffer produced measurable amounts of 4-nitrophenol after incubation for 60 min at 37 degrees C. Reactions were terminated by the addition of 200 mL of 200 mM borate buffer ( pH 9.8). Conclusions: The assay is sufficiently sensitive for small volumes of synovial fluid, and is useful for the clinical diagnosis of joint diseases.