Fluorescence Differentiation of ATP-related Multiple Enzymatic Activities in Synovial Fluid as a Marker of Calcium Pyrophosphate Deposition Disease using Kyoto Green.

Calcium pyrophosphate deposition disease (CPPD) is a crystal induced inflammation in joints, and causes severe pain in elderly people. The accumulation of pyrophosphate (PPi) in synovial fluid (SF) results from several enzymatic reactions, especially the highly activated e-NPPs, which catalyze the conversion of ATP to PPi. This study demonstrates the detection of relative catalytic activity of 3 enzymes-ecto-nucleotide pyrophosphatase/phosphodiesterases (e-NPPs), tissue nonspecific alkaline phosphatase (TNAP), and ecto-nucleoside triphosphate diphosphohydrolases (e-NTPDases)-using a single molecular sensor called Kyoto Green. Kyoto Green exhibits excellent performance in sensing the catalytic activity of the commercial representatives of the e-NPPs, TNAP, and e-NTPDases, which are ENPP1, PPase, and apyrase, respectively, in both single-enzyme and multi-enzyme assays. Analysis of SF enzymes in 19 SF samples from human and swine revealed moderate activity of e-NPPs, high activity of e-NTPDases, and low activity of TNAP. Our newly developed method for analysis of multiple enzymatic activities using Kyoto Green in biological SF will assist improvement in accuracy of the CPPD prognosis/diagnosis, which will minimize unnecessary medical procedures.

Low prevalence of knee chondrocalcinosis and its catabolic association with serum matrix metalloproteinase 3: A rural Japanese population study.

AIM: This study aimed to elucidate the prevalence of radiographic knee chondrocalcinosis (CC) and to clarify whether CC is correlated with self-reported knee symptoms and a serum catabolic biomarker. METHODS: A total of 1278 volunteers participated. Plain radiographs of both knees were obtained. Identification of a linear calcification in the knee joint space was defined as CC. Patients with a Kellgren-Lawrence grade of 2 or more were considered to have knee osteoarthritis (OA). Symptoms were evaluated using the Knee injury and Osteoarthritis Outcome Score (KOOS) Pain scale, and serum matrix metalloproteinase-3 (MMP-3) concentration was determined. Multiple regression analysis was conducted to determine whether CC was correlated with OA, the KOOS Pain scale and MMP-3 concentration. RESULTS: Twenty-eight subjects were found to have CC (2.2%), and 389 had OA (30.4%). CC was correlated with OA (odds ratio: 5.797; P = 0.006). Additionally, CC was correlated with MMP-3 concentration (B = 11.415, ß = 0.059, P = 0.014), but not with KOOS Pain scale. CONCLUSIONS: The prevalence of CC was low in the Japanese population evaluated in this study. While CC was not correlated with self-reported knee symptoms, it was positively correlated with serum MMP-3 concentration.

Ankylosing spondylitis, late osteoarthritis, vascular calcification, chondrocalcinosis and pseudo gout: toward a possible drug therapy.

In this review we consider diseases associated with pathological mineralization/ossification, namely, ankylosing spondylitis (AS), osteoarthritis (OA), generalized artery calcification of infancy (GACI), vascular calcification as well as chondrocalcinosis (CC) and pseudo gout. Deciphering the key enzymes implicated in the calcification process is an objective of prime importance and the ultimate goal is to synthesize inhibitors of these enzymes in order to provide efficient alternate therapeutic strategies that will slow down the pathologic mineralization and complement the arsenal of anti-inflammatory drugs. One of the difficulties in the definition of diseases associated with pathologic mineralization/ossification lies in the controversial relationship between the type of calcification and the nature of the disease. Here, we propose to clarify this relationship by making a distinction between diseases associated with hydroxyapatite (HA) and calcium pyrophosphate dihydrate (CPPD) deposits. AS, OA, GACI and vascular calcification are usually characterized by mineralization/ossification associated with HA deposits, while CC and pseudo gout are mostly characterized by CPPD deposits. Although both HA and CPPD deposits may occur concomitantly, as in chronic pyrophosphate arthritis or in OA with CPPD, they are formed as a result of two antagonistic processes indicating that treatment of distinct diseases can be only achieved by disease-specific drug therapies. The hydrolysis of PPi, an inhibitor of HA formation, is mostly controlled by tissue non-specific alkaline phosphatase TNAP, while PPi production in the extracellular medium is controlled by ANK, a PPi transporter, and/or NPP1 which generates PPi from nucleotide triphosphates. Low PPi concentration may lead to a preferential deposition of HA while high PPi concentration will favor the formation of CPPD deposits. Thus, HA and CCPD deposition cannot occur concomitantly because they are determined by the Pi/PPi ratio which, in turn, depends on the relative activities of antagonistic enzymes, TNAP hydrolyzing PPi or ANK and NPP1 producing PPi. TNAP inhibitors could prevent HA formation in AS, in late OA, in GACI, as well as in vascular calcifications, while ANK or NPP1 inhibitors could slow down CCPD deposition in CC and pseudo gout.

Inhibition of alkaline phosphatase by cysteine: implications for calcium pyrophosphate dihydrate crystal deposition disease.

OBJECTIVE: Calcium pyrophosphate dihydrate (CPPD) crystal deposition disease, a common arthritis affecting the elderly, is characterized by the deposition of CPPD crystals in articular joints. The mechanism underlying disease expression is unknown, but factors contributing to the pathogenesis may involve changes in enzymatic activities involving pyrophosphate and phosphate metabolism. Tissue nonspecific alkaline phosphatase (TNAP) is one of the major enzymes regulating pyrophosphate concentrations in articular joints. We hypothesized that inhibition of TNAP activity at pH = 7.4 by endogenous molecules can lead to CPPD disease pathogenesis. METHODS: We investigated the inhibitory effects of the amino acid cysteine on TNAP's phosphatase, inorganic pyrophosphatase, and CPPD crystal dissolution activities. Kinetic parameters V(max), K(M), concentration for 50% inhibition (I(50)), inhibitor constant (K(I)), and specific activities calculated from Initial Velocity, Eadie-Hofstee, Simple, Dixon, and Secondary plots were used to assess enzyme inhibition. RESULTS: Cysteine inhibited TNAP's phosphatase activity uncompetitively and its inorganic pyrophosphatase activity mix-competitively. CPPD crystal dissolution activity was also inhibited. I(50) values demonstrated that high cysteine concentration is required to inhibit 50% of enzyme activity. K(I) values suggested that inorganic pyrophosphatase activity is inhibited more than the phosphatase activity. Ca(++) and Mg(++) ion concentrations may regulate this inhibition. CONCLUSION: The control of endogenous inhibitors, such as cysteine, that interfere with TNAP's ability to regulate CPPD crystal formation and dissolution in joints could be a potential therapeutic option for CPPD crystal deposition disease.

MMP protein and activity levels in synovial fluid from patients with joint injury, inflammatory arthritis, and osteoarthritis.

OBJECTIVE: To determine protein and activity levels of matrix metalloproteinases 1 and 3 (MMP-1 and MMP-3) in synovial fluid of patients with knee joint injury, primary osteoarthritis, and acute pyrophosphate arthritis (pseudogout). METHODS: Measurements were done on knee synovial fluid obtained in a cross sectional study of cases of injury (n = 283), osteoarthritis (n = 105), and pseudogout (n = 65), and in healthy controls (n = 35). Activity of MMP-1 and MMP-3 in alpha(2) macroglobulin complexes was measured using specific low molecular weight fluorogenic substrates. ProMMP-1, proMMP-3, and TIMP-1 (tissue inhibitor of metalloproteinase 1) were quantified by immunoassay. RESULTS: Mean levels of proMMP-1, proMMP-3, and TIMP-1 were increased in injury, osteoarthritis, and pseudogout compared with controls. MMP-1 activity was increased in pseudogout and injury groups over control levels, whereas MMP-3 activity was increased only in the pseudogout group. The increase in MMP-1 activity coincided with a decrease in TIMP-1 levels in the injury group. CONCLUSIONS: Patients with joint injury have a persistent increase in proMMP-1 and proMMP-3 in synovial fluid and an increase in activated MMPs, which are not inhibited by TIMP. The differences in activation and inhibition patterns between the study groups are consistent with disease specific patterns of MMP activation and/or inhibition in joint pathology.

Extracellular nucleotides, cartilage stress, and calcium crystal formation.

Nucleotides are released by chondrocytes at rest and in response to mechanical stimulation. Extracellular nucleotides are metabolized by a variety of ectoenzymes, producing free phosphate (Pi) or pyrophosphate (PPi) and promoting matrix mineralization. Ectoenzymes are differentially localized in cartilage and may be co-released with nucleotides during mechanical stimulation. Extracellular nucleotides can also serve as substrates and/or modulators of enzymes such as tissue transglutaminase and ecto-protein kinases that modify matrix proteins and regulate crystal deposition or growth. Understanding the evolution of osteoarthritis and calcium crystal deposition diseases will require clearer knowledge of the functions of nucleotides and ectoenzymes in the cartilage extracellular matrix.

The nucleoside triphosphate pyrophosphohydrolase isozyme PC-1 directly promotes cartilage calcification through chondrocyte apoptosis and increased calcium precipitation by mineralizing vesicles.

OBJECTIVE: Aging associated elevations of cartilage extracellular inorganic pyrophosphate (PPi) and PPi-generating nucleoside triphosphate pyrophosphohydrolase (NTPPPH) are linked with degenerative arthritis in chondrocalcinosis. Increased chondrocyte apoptosis and expression of annexin V occur at sites of matrix calcification in degenerative arthritis, and membrane limited chondrocyte apoptotic bodies containing NTPPPH may promote chondrocalcinosis by acting as mineralizing matrix vesicles (MV). Because chondrocytes express 3 related NTPPPH isozymes [PC-1, autotaxin (ATX), and B10/PDNP3], we evaluated the effects on apoptosis and MV mediated calcium precipitation of direct expression of NTPPPH isozymes. METHODS: To achieve "gain of function" of NTPPPH isozymes, we expressed the isozymes in cultured chondrocytic cells. RESULTS: Plasmid cDNA transfection of PC-1, but not ATX or B10/PDNP3, markedly increased apoptosis of cultured chondrocytic knee meniscal cells and increased calcium precipitation by MV fractions. The capacity of PC-1 to increase chondrocyte and meniscal cell apoptosis, and calcium precipitation by MV, further analyzed using adenoviral gene transfer in cultured meniscal cells and articular chondrocytes, was shown to be dependent on integrity of the PC-I NTPPPH catalytic site. The MV-containing fraction released from meniscal cells and chondrocytes that overexpressed wild-type PC-1 had increased annexin V. Use of antibodies to annexin V and PC-1 revealed that both annexin V and PC-1 directly mediated the elevated calcium-precipitating capacity of MV. The increased ability of MV to precipitate calcium from PC-1-overexpressing cells did not require exogenous ATP. CONCLUSION: Upregulated expression of enzymatically active PC-1 directly promotes apoptosis, increased MV annexin V, and an increased capacity of meniscal cell and articular chondrocyte MV to precipitate calcium. These results suggest a direct link between increased PC-1 expression and the pathogenesis of chondrocalcinosis.

Serum nucleotide pyrophosphohydrolase activity; elevated levels in osteoarthritis, calcium pyrophosphate crystal deposition disease, scleroderma, and fibromyalgia.

OBJECTIVE: Quantification of serum nucleotide pyrophosphohydrolase (NTPPHase) activity in healthy subjects and in patients with various rheumatic diseases or with quad/hemiplegia, hemodialysis, or renal transplant. METHODS: Colorimetric assay of enzyme activity in serum. RESULTS: Serum NTPPHase activity in 85 healthy subjects was independent of age or sex and was highly reproducible in each individual. The biologic and methodologic coefficients of variation were nearly identical. Elevated enzyme levels were found in sera from patients with osteoarthritis/spondylosis, calcium pyrophosphate dihydrate (CPPD) crystal deposition, scleroderma, fibromyalgia, or hemodialysis. Renal transplant patients receiving cyclosporine had the highest enzyme activity of any group, whereas transplant patients not taking this drug had normal levels. Histograms of values in all groups showed a normal distribution. CONCLUSION: Serum NTPPHase activity levels were significantly elevated in patients with degenerative arthritis whether or not CPPD crystals were present, in patients with either scleroderma or fibromyalgia, and in patients receiving hemodialysis therapy or taking cyclosporine.

Regulation of alkaline phosphatase: implications for calcium pyrophosphate dihydrate crystal dissolution and other alkaline phosphatase functions.

OBJECTIVE: Alkaline phosphatase (ALP), an enzyme with pyrophosphatase (PPiase) activity can dissolve calcium pyrophosphate dihydrate (CPPD) crystals. We studied the effects of enzyme inhibitors such as bisphosphonates, orthovanadate, calcium, cadmium, and ascorbic acid on PPiase activity of ALP as well as on phosphate ester hydrolysis (Pase) activity and compared these effects to those on CPPD crystal dissolution. METHOD: An in vitro model system for crystal enzyme interaction was used to assess CPPD crystal dissolution. RESULTS: Bisphosphonates inhibited ALP Pase activity more than ALP PPiase activity at the same concentrations. Calcium inhibited ALP PPiase activity, but not ALP Pase activity. Orthovanadate and cadmium inhibited ALP PPiase activity more than ALP Pase at the same concentrations. The inhibition rates of ALP PPiase at the same concentrations were orthovanadate > cadmium > calcium. Although ALP Pase activity was not inhibited, at high concentrations, ascorbic acid slightly inhibited ALP PPiase activity. Bisphosphonates at high concentrations inhibited ALP CPPD crystal dissolution. The strong inhibitory effects of bisphosphonates on ALP CPPD crystal dissolution compared to those on ALP PPiase activity suggest that bisphosphonates inhibit crystal dissolution by their affinity for the CPPD crystal surface. Calcium, orthovanadate, and cadmium inhibited ALP CPPD dissolution. The inhibition rates of ALP CPPD dissolution at the same concentrations were cadmium > calcium > orthovanadate. Ascorbic acid at high concentrations enhanced ALP CPPD dissolution. CONCLUSION: These effects of different inhibitors on ALP PPiase and CPPD dissolution suggest that ALP CPPD crystal dissolution depends on binding of ALP CPPD crystals as well as the PPiase activity of the bound ALP. Because of its ubiquitous and broad phosphatase activity including PPiase activity, ALP may have a critical role in cell energy metabolism.

Adenosine triphosphate pyrophosphohydrolase and neutral inorganic pyrophosphatase in pathologic joint fluids. Elevated pyrophosphohydrolase in calcium pyrophosphate dihydrate crystal deposition disease.

Adenosine triphosphate pyrophosphohydrolase (ATPPPH) and neutral inorganic pyrophosphatase activities were assayed in synovial fluids (SF) from 37 patients with a variety of arthropathies. ATPPPH activity was detected in all fluids, but was highest in patients with chronic chondrocalcinosis; its activity in patients with osteoarthritis was higher than that in patients with rheumatoid arthritis, gout, or pseudogout. ATPPPH activity correlated positively with SF pyrophosphate concentration and negatively with SF white blood cell count. Pyrophosphatase activity did not correlate with diagnosis, pyrophosphate level, or white blood cell count.

[Non-lymphoid mononucleated cells in the synovial fluid in arthrosis and various inflammatory arthropathies. Morphologic, quantitative and cytoenzymologic study]. / Les cellules mononucléées non lymphoïdes du liquide synovial dans l'arthrose et diverses arthropathies inflammatoires. Etude morphologique, quantitative et cyto-enzymologique.

This paper describes a morphologic, quantitative, cytochemical study of mononuclear non lymphoid cells in knee synovial fluid in osteoarthritis and various arthritides. Morphologic criteria allow to identify among these cells various synoviocytic and monocytic subtypes with in both types, phagocytic subtypes. Quantitative study shows in arthritides an important afflux of monocytes and a hyperexfoliation of synoviocytes. In fluids with intermediate cellularity, Monocytes/Synoviocytes ratio allows the differential cytodiagnosis between osteoarthrosis and arthritis. All monocytic subtypes and especially the phagocytic one are highly significantly increased in arthritides. Synoviocytic subtypes show a lower increase, except the phagocytic one, which is not changed. Giant multinuclear synoviocytes are found in every type of disease and cannot constitute a cytodiagnosis marker. Alcian Blue and hyaluronidase treatment show hyaluronate in a few percentage of Synoviocytes. Cytoenzymologic study shows that synoviocytes and monocytes are positive in all tested hydrolases: beta Glucuronidase, Acid Phosphatase, alpha Naphthyl Acetate Esterase, these activities being always higher in synoviocytes. With peroxidase, synoviocytes are always negative, so this reaction although it marks only a minority of monocytic population can be used as an extra cytologic criterion for discrimination of mononuclear cells in synovial fluid. In these four enzymes there is no significant quantitative difference at cellular level between osteoarthrosis and arthritides. Lysosomal enzymatic activity in both monocytic and synoviocytic cells confirms their heterophagic properties. However synoviocytic heterophagy seems to be a physiological process not or few affected by inflammatory events. On the opposite, monocytic heterophagy and then macrophagic transformation of monocytes appears as a major aspect of intrasynovial inflammatory reaction. If a large majority of exfoliated synoviocytes comes from A type synovial lining cells and if they belong to Mononuclear Phagocyte System, why do they so weakly, or not, participate as phagocytes to inflammatory reaction.

NTP pyrophosphohydrolase in human chondrocalcinotic and osteoarthritic cartilage. II. Further studies on histologic and subcellular distribution.

A nucleoside triphosphate (NTP) pyrophosphohydrolase was previously demonstrated in human chondrocalcinotic and osteoarthritic articular cartilage. In this study, grinding and enzymatic techniques for cartilage subcellular fractionation were compared. It was shown that this enzyme was concentrated in fractions enriched in plasma membranes. The enzyme had the same activity in cartilage slices as in triton X-100 extracts, and all of the activity was removed by preliminary treatment of cartilage slices with pronase. Histologic separation of cartilages into tangential and upper radial versus lower radial zones indicated no difference in concentrations of this enzyme. A role is postulated for this enzyme in metabolic chondrocytic plasma membrane responses to injury or destabilization from other causes, such as rapid synthesis or cell division.

NTP pyrophosphohydrolase in human chondrocalcinotic and osteoarthritic cartilage. I. Some biochemical characteristics.

Nucleoside triphosphate pyrophosphohydrolase activity was first detected in articular cartilage in previous studies at our laboratory. In this report, the enzyme is partially characterized with respect to its pH optimum and Km. The enzyme was metal-dependent and was active in the presence of 1 mM Ca++. It was inhibited by several substances, including cysteine and dithiothreitol. Its activity was not inhibited by tetramisole at concentrations which inhibited 100% of the pyrophosphatase activity in the same extracts. It functioned most effectively on ATP, but also on UTP, CTP, and GTP. A role for scavenging nucleotides and production of pyrophosphate in osteoarthritic and chondrocalcinotic cartilage is postulated.

Inorganic pyrophosphatase activity of the synovial fluid. Kinetic and clinical study.

Inorganic pyrophosphatase activity has been partially characterized in joint fluid and determined in patients with sporadic and familial chondrocalcinosis and their controls. Optimal pH was established at 3.5 and Km values were estimated. Ca-2 and Mg-2 did not affect this activity whereas orthophosphate (Pi) strongly inhibited it. In the clinical study no significant differences were found among groups. This suggests that if a pyrophosphatase defect is present it might be localized in joint tissue and not be reflected in synovial fluid.

N-acetyl-beta-D-hexosaminidase system in synovial fluid.

The present study was undertaken with the object of examining the N-Acetyl-beta-D-hexosaminidase activity in joint effusions from non-inflammatory (osteoarthrosis) and inflammatory (rheumatoid arthritis, gouty arthritis and chondrocalcinosis) joint diseases. The biochemical properties of four purified molecular forms were investigated. They were separated on the basis of their net charge, using DEAE-cellulose anion-exchangers. In the order of their outcome from the anion exchanger the four enzymes (B, I1, I2 and A) were found to have pH optima at 4.5-4.75, 4.20, 4.00 and 4.75, respectively. The first three enzymes proved to be heat-stable and the fourth enzyme fraction was a heat-labile form. By means of gel-filtration techniques, the enzymes were eluted into two fractions. The first contained the thermolabile A form and the molecular weight of this enzyme was estimated at 162000. The second fraction included the three thermostable enzymes. Their molecular weight was estimated at 135000. As described by Ikonne & Ellis (6) the hexosaminidase A from sera was less tightly held by the anion-exchanger than was the hexosaminidase A from tissues (polymorphonuclear cells, synovial membrane tissue, cartilage). Thus the serum type (As) must be distinct from the tissue type (At). The activity of the tissue type probably originating from the leukocytes and from the synovial membrane was more pronounced in the synovial effusions of the patients with inflammatory joint diseases. The hexosaminidase A fraction from synovial fluids of patients with osteoarthrosis contained only the serum type of enzyme.

[Importance of enzymes in the synovial fluid. Progress report]. / Importanza degli enzimi nel liquido sinoviale. Nota di aggiornamento

The significance and importance of investigation of the synovial fluid enzymes in the main arthropathies are explanined. Tables are given for the main enzymes studied, the cell compartments of origin, and data for their values in rheumatic diseases (as reported in the literature). Stress is laid on the importance of enzymes belonging to the lysosomial compartment, both in the pathogenesis of the underlying inflammation and in the relation to anatomopathological lesions in the joints. Attention is directed to the most widely accepted hypotheses. These ten to see enzymes increases in breakdown of condrocytes, as inflammatory arthritis attributable to synoviocytes and leukocytes. A personal opinion based on prior research is also presented. Further work in this sector is urged a mean of learning more about the pathology of rheumatic diseases.

N-acetyl-beta-d-glucosaminidase activity in synovial fluid.

N-acetyl-beta-D-glucosaminidase activity in the synovial fluid of different articular diseases was studied and statistical investigations were carried out after logarithmic transformation of the data. The enzyme activity in the synovial fluid of rheumatoid arthritis is increased when compared with osteoarthrosis and traumatic effusions. The enzyme activity in traumatic effusions is also increased in comparison with osteoarthrosis. A linear relation was found between the enzyme activity in cell-free fluid and the polymorphonuclear leucocyte (PMN) count in rheumatoid arthritis osteoarthrosis and in miscellaneous synovitis. The activity per PMN cell was determined and found to be relatively constant in the synovial fluid of inflammatory diseases (rheumatoid arthritis, chondrocalcinosis, miscellaneous synovitis). The N-acetyl-beta-D-glucosaminidase activity per PMN cell in serum was found to be significantly lower than in synovial fluid.