NUCLEOTIDE PYROPHOSPHOHYDROLASE IN HUMAN SYNOVIAL-FLUID

Objective. To identify the molecular forms of ectonucleotide pyrophosp hohydrolase (NTPPHase) in human synovial fluid (SF). Methods, We exami ned synovial fluids from 32 patients with various joint diseases [10 c alcium pyrophosphate dihydrate (CPPD) deposition disease; 7 osteoarthr itis (OA); 6 rheumatoid arthritis (RA); 3 after total knee arthroplast y (TKA); 6 olecranon bursa] and 3 normal joint fluids. Joint fluids we re analyzed after sequential centrifugation for NTPPHase activity and by Western blot using polyclonal antibodies against 127 kDa porcine ar ticular cartilage vesicle-associated NTPPHase and against PC-I and 58 kDa, 2 other ecto-NTPPHases. Lysate from human synoviocytes, porcine c hondrocytes, and their conditioned media were examined using antibodie s to these ecto-NTPPHases. Radiographs of joints from which fluid was obtained were graded for degenerative changes 0-4 using a standard met hod. Results, NTPPHase activity was found in all pathological and norm al SF tested and correlated with the degree of radiographic degenerati on (r = 0.55, p < 0.05). NTPPHase specific activity in ultracentrifuga tion pellets was highest in CPPD deposition disease fluids !p < 0.05). 127 kDa enzyme was found in both sedimentable and soluble fractions f rom CPPD, OA, TKA, and normal fluids, and was extensively degraded in all inflammatory fluids. Intact 115 kDa PC-I was found only in the 2 C PPD fluids with the highest NTPPHase activity. 58 kDa enzyme was found in most fluids, predominantly in the soluble fraction. 127 kDa protei n was identified in human synoviocyte conditioned media but not in cel l lysate, while PC-I and 58 kDa proteins were found in the cell lysate but not in the conditioned media. Conclusion. There was no disease sp ecific association with any one ecto-NTPPHase. Total enzyme activity c orrelated with the degree of degenerative change. The specific activit y of pelletable 127 kDa enzyme was higher in fluids containing CPPD cr ystals. All 3 ecto-NTPPHases or their presumed degradation products we re detectable in some pathologic and normal fluids. A 200 kDa reactive band often accompanied reactivity to the 127 kDa enzyme. PC-1 and 127 kDa proteins were extensively degraded in inflammatory SF, while 58 k Da protein was not. The relative contribution of each of these enzymes to inorganic pyrophosphate production by human joint tissues remains unclear.