Abstract

Background: Our goal was to develop a suitably sensitive assay for N-acetyl-β-D-hexosaminidase (HEX) and β-glucuronidase to allow their use as markers of joint diseases.

Methods: We optimized a spectrophotometric method for the determination of lysosomally derived HEX and β-glucuronidase in synovial fluid on a microplate reader to improve its utility. HEX and β-glucuronidase act on the 4-nitrophenyl derivatives N-acetyl-β-glucosamine and β-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 β-glucuronidase. A 10-μL sample with 30 μL of substrate solution and 40 μL of appropriate buffer produced measurable amounts of 4-nitrophenol after incubation for 60 min at 37°C. Reactions were terminated by the addition of 200 μL 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.

Clin Chem Lab Med 2006;44:933–7.