The synovial fluid concentration of cartilage """"""""markers"""""""", e.g., portions of the proteoglycan (PG) molecule of proteins derived from articular cartilage matrix, has been used to deduce the magnitude of cartilage destruction or repair in osteoarthritic joints. However, because the kinetics of marker removal from the joint are uncertain in such studies, they can not provide a quantitative measure of cartilage metabolism. As shown in our laboratory, variables that change the clearance kinetics of synovial fluid macromolecules, such as the synovitis which is often present in osteoarthritis (OA), must be taken into account if conclusions about AC metabolism are to be based on changes in the concentration of a synovial fluid """"""""marker."""""""" Synovial fluid macromolecules exit the joint through the lymphatics, and their concentration is inversely related to the rate of lymphatic outflow from the joint. Because there is evidence that the molecular weight (MW) of a protein does not affect its rate of clearance from the joint space, the clearance kinetics of 131I-labeled albumin (RISA) have been used to estimate the clearance of """"""""marker"""""""" proteins from the joint. There is no direct evidence, however, that RISA and larger proteins, e.g., cartilage oligomeric matrix protein (COMP), or the proteoglycan, aggrecan, a """"""""marker"""""""" molecule heavily substituted with sulfated glycosaminoglycans (GAG), are cleared at the same rate as RISA. Notably, hyaluronan (HA), the principal GAG in synovial fluid, is cleared from the joint substantially more slowly (t1/2=12-14 hrs) than RISA (t1/2=4hrs) and form strong, noncovalent bonds with PG and link protein (LP). Limited data suggest that the t1/2 of PG in the joint is approximately 12 hrs, and thus resembles the value for HA. The hypothesis tested in this study is that the reduced clearance of a """"""""marker"""""""" which leaves the joint more slowly than albumin is related to its capacity to bind HA, and to its MW. PG, COMP, LP and fragments of PG which retain the capacity to interact with HA will be isolated from normal articular cartilage. These molecules, and albumin, myosin and beta-galactosidase, as surrogates for plasma-derived synovial fluid proteins, will be radiolabeled with 131I, and then mixed with high MW HA. RISA will be injected into the right knee of normal rabbits, and its clearance will be determined by monitoring radioactivity at the joint surface, and aspiration of synovial fluid. One week later, clearance of one of the 131I-cartilage-derived molecules will be determined in the contralateral knee in exactly the same manner. We will: 1) establish whether the rate of clearance of aggrecan whose HA-binding region (HABR) is intact differs from that of RISA, or of PG lacking HABR, 2) assess the clearance kinetics of COMP (MW = 500 kDa), myosin (MW = 200 kDa), beta-galactosidase (MW =116 kDa) and RISA (MW = 66 kDa) to relate the MW of each protein to its rate of removal from the joint, and 3) contrast the clearance kinetics of cartilage-derived proteins whose MW resemble that of RISA, but which can bind synovial fluid HA, i.e., fragments of PG and LP, to those of RISA and PG fragments that are unable to interact with HA, and thereby assess whether interactions between these molecules and HA influence their removal from the joint and concentration in synovial fluid.
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