Inorganic pyrophosphate generation from adenosine triphosphate by cell-free human synovial fluid
Park, W.; Masuda, I.; Cardenal-Escarcena, A.; Palmer, D.L.; McCarty, D.J.
Journal of Rheumatology 23(4): 665-671
1996
ISSN/ISBN: 0315-162X PMID: 8730124 Document Number: 466998
Objective. To quantify inorganic pyrophosphate (PP-i) production from extracellular adenosine triphosphate (ATP) by human synovial fluids (SF). Methods. Serial measurements of ATP hydrolysis rate (t1/2) were performed by the luciferase method from a starting concentration of 500 nM in 21 pathologic and one normal cell-free SF samples incubated under physiologic conditions. ATP was then pumped into a sample of each fluid, using the rate constant derived from the t1/2 of that fluid, to provide steady state levels simulating those reported in SF. Trace (32P) gamma ATP was added at the start of the infusion; conversion to (32P) P-i and to (32P) PP-i was determined by precipitation of P-i as reduced phosphomolybdate before and after treatment with yeast inorganic pyrophosphatase. Finally, the pumping experiment was repeated and PP-i production was calculated from direct measurement of PP-i at time zero and at 60 min. PP-i hydrolysis was measured in each fluid by (32P) P-i precipitation from (32P) PP-i tracer added at time zero. Results. ATP was hydrolyzed by all SF. The mean t1/2 (seconds) in 8 osteoarthritis (OA) samples was 72 s, in 5 calcium pyrophosphate dihydrate (CPPD) 30 s (p lt 0.02), in 3 rheumatoid arthritis (RA) 1160 s, in one normal 86 s, in 3 olecranon bursal (OB) 54 s, and in 2 total knee replacement fluid samples 17 and 121 s. The major product of ATP hydrolysis was PP-i in all but 2 fluids (1 RA, 1 OB), even at lower than steady state levels. At simulated in vivo steady state ATP levels, mean conversion of ATP to PP-i was stoichiometric in OA and CPPD fluids. PP-i hydrolysis was lt 4% in all noninflammatory fluids. Conclusion. PP-i is the major product of extracellular ATP catabolism in most SF. Hydrolysis rates were significantly faster in SF containing CPPD crystals. Mean PP-i production by these fluids at simulated in vivo steady state levels was 6-fold that of OA SF (p lt 0.01). Hydrolysis of extracellular ATP by ectonucleotide pyrophosphohydrolases can account for all PP-i produced by joint tissues previously estimated from (32P) PP-i pool and turnover studies in human knee joints.