**7. Assay of exoglycosidase activity**

Samples of the synovial fluid are easily taken from the knee joints during diagnostic or therapeutic arthrocentesis or at arthroscopy. Synovial fluid is aspirated from a lateral infrapatellar approach with a 21G needle (Fig. 10).

Fig. 9. The specific activity of HEX in synovial fluid of patients with rheumatoid arthritis.

from the same patients. This results suggest that HEX in synovial fluid derives mainly from articular tissues or articular leukocytes and not from serum. Therefore, activity of HEX in synovial fluid better reflects the situation in the joint cavity than activity of HEX

The differences between specific activities of HEX (activities calculated per 1 kg of protein) in synovial fluid of RA and JIA patients in comparison to OA and ACL patients (Fig. 9) are even more evident than differences in concentration of HEX (activities calculated per

Fig. 9. The specific activity of HEX in synovial fluid of patients with rheumatoid arthritis.

Significant increase of specific HEX activity in synovial fluid of JIA patients (in comparison to OA and control) may be of diagnostic value in children with prolonged exudates in the knee joint, who are resistant to pharmacological and physiotherapeutical treatment. In these cases we advise determining specific activity of HEX in the synovial fluid, where values above 10-13 µkat/kg of protein suggest rheumatoid disease. It is worthy of note that specific activity of HEX in synovial fluid of patients with RA demonstrates a broad standard deviation which probably depends on destructive or inflammatory processes in the joint. However, significantly elevated HEX activity indicated an inflammatory or autoimmunological process

Samples of the synovial fluid are easily taken from the knee joints during diagnostic or therapeutic arthrocentesis or at arthroscopy. Synovial fluid is aspirated from a lateral

in serum.

within the joint.

**7. Assay of exoglycosidase activity** 

infrapatellar approach with a 21G needle (Fig. 10).

volume of synovial fluid) in the same situations.

Fig. 10. Synovial fluid aspiration from the knee.

To obtain reliable results of exoglycosidase determinations, patients with inflammatory arthritis should avoid steroid drug treatment at the time of arthrocentesis, and for two days before arthrocentesis. About 1 ml of synovial fluid is sufficient for assays of exoglycosidase activity and protein concentration. Samples of synovial fluid are collected in plastic tubes and centrifuged at 10,000x g for 30 min, separated from the cell pellet, and stored at -70º C before use. As substrates for exoglycosidase activity we use p-nitrophenyl derivatives of appropriate sugars purchased from Sigma, St.Louis, Mo, USA (HEX, GAL, MAN, FUC), and Fluka Chemie GmbH (GluA).

The activities of HEX (E.C. 3.2.1.52), β-glucuronidase (E.C. 3.2.1.31), β-galactosidase (E.C. 3.2.1.23), α-mannosidase (E.C. 3.2.1.24), and α-fucosidase (E.C. 3.2.1.51) are determined by simple and inexpensive methods (Marciniak et. al. 2006). Before exoglycosidases determinations, the samples of synovial fluid are diluted with 0.1 M of the appropriate buffer and incubated with excess of substrate for 60 min at 37º C. The reaction is stopped by adding 0.2 M borate buffer, pH 9.8. HEX A activity is calculated as the difference between the total HEX activity and HEX B activity.

Spectrophotometric measurements of released 4-nitrophenol were carried out at 405 nm using a microplate reader Elx800TM. The concentration of enzymatic activity of the appropriate exoglycosidase was expressed as nanomoles of p-nitrophenol released per minute per ml in synovial fluid and specific activity was expressed in µkat/kg of proteins of synovial fluid. The concentration of exoglycosidase activity indicates the ability of the specified volume of synovial fluid to release the quantity of monosaccharide indicated. The specific activity relates a particular exoglycosidase activity to total protein concentration, and shows the proportion of exoglycosidase protein to the total protein content of an articular sample of synovial fluid.

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