INORGANIC PYROPHOSPHATE GENERATION FROM ADENOSINE-TRIPHOSPHATE BY CELL-FREE HUMAN SYNOVIAL-FLUID

Objective, To quantify inorganic pyrophosphate (PP,) production from e xtracellular adenosine triphosphate (ATP) by human synovial fluids (SF ). Methods. Serial measurements of ATP hydrolysis rare (t(1/2)) were p erformed by the luciferase method from a starting concentration of 500 nM in 21 pathologic and one normal cell-free SF samples incubated und er physiologic conditions. ATP was then pumped into a sample of each f luid, using the rate constant derived from the t(1/2) of that fluid, t o provide steady state levels simulating those reported in SF. Trace [ P-32] gamma ATP was added at the start of the infusion; conversion to [P-32] P-i and to [P-32] PPi was determined by precipitation of P-i as reduced phosphomolybdate before and after treatment with yeast inorga nic pyrophosphatase. Finally, the pumping experiment was repeated and PPi production was calculated from direct measurement of PPi at time z ero and at 60 min. PPi hydrolysis was measured in each fluid by [P-32] P-i precipitation from [P-32] PPi tracer added at time zero. Results. ATP was hydrolyzed by all SE The mean t(1/2) (seconds) in 8 osteoarth ritis (OA) samples was 72 s, in 5 calcium pyrophosphate dihydrate (CPP D) 30 s (p < 0.02), in 3 rheumatoid arthritis (RA) 1160 s, in one norm al 86 s, in 3 olecranon bursal (OB) 54 s, and in 2 total knee replacem ent fluid samples 17 and 121 s. The major product of ATP hydrolysis wa s PPi in all but 2 fluids (1 RA, 1 OB), even at lower than steady stat e levels. At simulated in vivo steady state ATP levels, mean conversio n of ATP to PPi was stoichiometric in OA and CPPD fluids. PPi hydrolys is was <4% in all noninflammatory fluids. Conclusion. PPi is the major product of extracellular ATP catabolism in most SE Hydrolysis rates w ere significantly faster in SF containing CPPD crystals. Mean PPi prod uction by these fluids at simulated in vivo steady state levels was 6- fold that of OA SF (p<0.01). Hydrolysis of extracellular ATP by ectonu cleotide pyrophosphohydrolases can account for all PPi produced by joi nt tissues previously estimated from [P-32] PPi pool and turnover stud ies in human knee joints.