Optimization of an enzymatic method for the determination of lysosomal N-acetyl-beta-D-hexosaminidase and beta-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 beta-glucuronidase. A 10-microL sample with 30 microL of substrate solution and 40 microL of appropriate buffer produced measurable amounts of 4-nitrophenol after incubation for 60 min at 37 degrees Celsius. Reactions were terminated by the addition of 200 microL 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.
Marciniak
Justyna
J
Department of Pediatric Orthopedics and Traumatology, Medical University, Białystok, Poland. jmarciniak@vp.pl
Zalewska
Anna
A
Popko
Janusz
J
Zwierz
Krzysztof
K
D000368:Aged; D001172:Arthritis, Rheumatoid / Q000201:enzymology*; D015415:Biomarkers / Q000378:metabolism*; D005260:Female; D005966:Glucuronidase / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000201:enzymology*; D008297:Male; D008875:Middle Aged; D013053:Spectrophotometry / Q000379:methods; D013379:Substrate Specificity; D013582:Synovial Fluid / Q000201:enzymology*; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism*
D015415:Biomarkers; D005966:Glucuronidase; D001619:beta-N-Acetylhexosaminidases