**6. Activity of lysosomal exoglycosidases in serum and synovial fluid of patients with RA and JIA**

Information concerning the activity of exoglycosidases in patients with joint diseases is ambiguous and limited to a few, mostly old, publications (Bartholomew 1972; Stephens et al.1975; Ganguly et al. 1978; Berenbaum et al. 2000; Sohar et al. 2002). Serum HEX activity was higher in 35 % of the RA patients than in healthy controls (Berenbaum et al. 2000). Berenbaum et.al. (2000) found that the serum HEX concentration was significantly higher in destructive RA than in inflammatory RA. Since RA patients with high serum HEX activity have more erosions, than those with inflammatory RA, but have no differences in CRP levels, it is possible that HEX is a marker of erosions.

Lysosomal enzymes in human polymorphonuclear leukocytes are ubiquitous, biologically active molecules, that can degrade macromolecules such as proteins, glycosaminoglycans, nucleic acids, and lipids (Sohar et al.2002). Sohar et.al. (2002) have suggested that the increase of HEX activity in the serum of RA patients may be caused by an increase in HEX

Fig. 7. Effect of interleukin 1β (IL-1β) on intra-(In) and extracellular (Ex) activity of lysosomal exoglycosidases in cultured human synovial cells from patients with anterior cruciate ligament (*ACL*) injuries, juvenile idiopathic arthritis (JIA), and rheumatoid arthritis (RA). Synoviocytes were stimulated with IL-1β for 24h at 37ºC. After IL-1β stimulation, cultured cells and exracellular fluid were analyzed for exoglycosidase activities. The effect of IL-1β was expressed as the enzymatic activity in stimulated versus unstimulated cultures. Rheumatoid synoviocytes exhibit altered morphology and show certain similarities to tumor cells (Mor et al. 2005). Our data confirmed the observation that synoviocytes obtained from patients with JIA and RA are more active in the synthesis and secretion of exoglycosidases than those obtained from patients with injured knees or healthy joints

**6. Activity of lysosomal exoglycosidases in serum and synovial fluid of** 

Information concerning the activity of exoglycosidases in patients with joint diseases is ambiguous and limited to a few, mostly old, publications (Bartholomew 1972; Stephens et al.1975; Ganguly et al. 1978; Berenbaum et al. 2000; Sohar et al. 2002). Serum HEX activity was higher in 35 % of the RA patients than in healthy controls (Berenbaum et al. 2000). Berenbaum et.al. (2000) found that the serum HEX concentration was significantly higher in destructive RA than in inflammatory RA. Since RA patients with high serum HEX activity have more erosions, than those with inflammatory RA, but have no differences in CRP

Lysosomal enzymes in human polymorphonuclear leukocytes are ubiquitous, biologically active molecules, that can degrade macromolecules such as proteins, glycosaminoglycans, nucleic acids, and lipids (Sohar et al.2002). Sohar et.al. (2002) have suggested that the increase of HEX activity in the serum of RA patients may be caused by an increase in HEX

(Popko et. al.2008). (Popko et. al.2008).

levels, it is possible that HEX is a marker of erosions.

**patients with RA and JIA** 

activity of RA leukocytes. Patients with long-standing RA had higher activity of lysosomal glycosidases in their leukocytes than those with disease of shorter duration.

We suggest that of the exoglycosidases, only HEX activity measurement in synovial fluid and serum has practical value (Popko et. al. 2006). The specific activity of HEX and its isoenzyme A in serum and synovial fluid from patients with different arthropathies is presented in Fig. 8.

Fig. 8. Specific activity of N-acetyl-β-hexosaminidase and its isoenzyme A in serum and synovial fluid (µkat/kg of protein).

In the serum of RA patients, the specific activity of HEX was significantly increased in comparison to control (Fig. 8). This increase of HEX activity in the serum of RA patients may depend on the increase in HEX activity of RA leukocytes (Sohar et al. 2002). In the serum of patients with JIA and OA, we observed a moderate increase in HEX activity, i.e. 25.46% and 17.3 % respectively. In the case of patients with ACL injury, the specific activity of HEX and its isoenzymes in serum behaved similarly as in the control.

In the synovial fluid of JIA and RA patients, we found a significant increase in the specific activity of HEX and its isoenzymes, in comparison to HEX activity in synovial fluid from OA and ACL injuries. This suggests that release of HEX to synovial fluid is greatly enhanced by the autoimmunological inflammatory process in knee joint cavity.

The activity of HEX in synovial fluid of patients with JIA and RA was significantly higher than the activity in OA and ACL patients. Additionally, we have found that in JIA and RA patients, the specific activity of HEX in synovial fluid is 6-8 times higher than in serum

Lysosomal Glycosidases in Degradation of Human Articular Cartilage 107

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

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

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

Fig. 10. Synovial fluid aspiration from the knee.

Fluka Chemie GmbH (GluA).

the total HEX activity and HEX B activity.

articular sample of synovial fluid.

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 in serum.

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 volume of synovial fluid) in the same situations.

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 within the joint.
