Park W, Masuda I, Cardenal-Escarcena A, Palmer D L, McCarty D J
Department of Medicine, Medical College of Wisconsin, Milwaukee 53226, USA.
J Rheumatol. 1996 Apr;23(4):665-71.
To quantify inorganic pyrophosphate (PPi) production from extracellular adenosine triphosphate (ATP) by human synovial fluids (SF).
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] Pi and to [32P] PPi was determined by precipitation of Pi as reduced phosphomolybdate before and after treatment with yeast inorganic pyrophosphatase. Finally, the pumping experiment was repeated and PPi production was calculated from direct measurement of PPi at time zero and at 60 min. PPi hydrolysis was measured in each fluid by [32P] Pi precipitation from [32P] PPi tracer added at time zero.
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 < 0.02), in 3 rheumatoid arthritis (RA) 1160 s, in 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 PPi 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 APT to PPi was stoichiometric in OA and CPPD fluids. PPi hydrolysis was < 4% in all noninflammatory fluids.
PPi is the major product of extracellular ATP catabolism in most SF. Hydrolysis rates were significantly faster in SF containing CPPD crystals. Mean PPi production 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 ectonucleotide pyrophosphohydrolases can account for all PPi produced by joint issues previously estimated from [32P] PPi pool and turnover studies in human knee joints.
定量测定人滑液（SF）从细胞外三磷酸腺苷（ATP）产生无机焦磷酸（PPi）的情况。
采用荧光素酶法，对21份病理样本和1份正常无细胞SF样本在生理条件下从500 nM的起始浓度进行ATP水解速率（t1/2）的系列测量。然后，利用从每种液体的t1/2得出的速率常数，将ATP泵入每种液体样本中，以提供模拟SF中报道水平的稳态水平。在输注开始时加入微量[32P]γ-ATP；在用酵母无机焦磷酸酶处理前后，通过将Pi沉淀为还原型磷钼酸盐来测定向[32P]Pi和[32P]PPi的转化。最后，重复泵送实验，并根据在时间为零和60分钟时对PPi的直接测量来计算PPi的产生量。通过在时间为零时加入的[32P]PPi示踪剂的[32P]Pi沉淀来测量每种液体中的PPi水解。
所有SF均可水解ATP。8份骨关节炎（OA）样本的平均t1/2（秒）为72秒，5份二水焦磷酸钙（CPPD）样本为30秒（p<0.02），3份类风湿关节炎（RA）样本为1160秒，正常样本为86秒，3份鹰嘴滑液（OB）样本为54秒，2份全膝关节置换液样本分别为17秒和121秒。除2份液体（1份RA、1份OB）外，所有液体中ATP水解的主要产物均为PPi，即使在低于稳态水平时也是如此。在模拟的体内稳态ATP水平下，OA和CPPD液体中APT向PPi的平均转化呈化学计量关系。所有非炎性液体中的PPi水解均<4%。
在大多数SF中，PPi是细胞外ATP分解代谢的主要产物。含CPPD晶体的SF中的水解速率明显更快。这些液体在模拟的体内稳态水平下的平均PPi产生量是OA SF的6倍（p<0.01）。外核苷酸焦磷酸水解酶对细胞外ATP的水解可解释先前根据人膝关节中[32P]PPi池和周转率研究估计的关节问题产生的所有PPi。
