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PubMed1995

Calcium pyrophosphate dihydrate (CPPD) crystal dissolution by alkaline phosphatase: interaction of alkaline phosphatase on CPPD crystals.

OBJECTIVE: As alkaline phosphatase (ALP) can dissolve calcium pyrophosphate dihydrate (CPPD) crystals, and as dissolution is facilitated when the enzyme is proximate to the crystals, we studied the mechanism of ALP interaction with CPPD crystals in vitro. METHODS: ALP was incubated with CPPD crystals in an in vitro model system. Fluorescein isothiocyanate conjugated alkaline phosphatase (FITC-ALP), alkaline phosphatase product staining of calcium pyrophosphate dihydrate (CPPD) crystals and scanning electron microscopy were used to visualize ALP-CPPD crystal interactions. RESULTS: ALP preferentially binds to the small end faces (optical 010 faces) of CPPD crystals. Etch pits indicative of dissolution were demonstrated coexistent with ALP crystal binding and ALP pyrophosphohydrolytic activity. CONCLUSION: ALP binding to CPPD crystals is preferential for the smallest end faces (optical 010 faces). As ALP crystal binding is altered by ions but not by heat inactivation of ALP, ALP-CPPD crystal binding is considered a nonenzymatic mechanism distinct from ALP pyrophosphohydrolytic activity. Our study demonstrates that ALP binds and dissolves CPPD crystals in a stereoselective manner. This suggests that the CPPD crystal dissolution rate is limited by the availability of surface area on the crystal faces most susceptible to ALP binding.

Alkaline Phosphatase↗
PubMed1994

Characterization of chondrocyte alkaline phosphatase as a potential mediator in the dissolution of calcium pyrophosphate dihydrate crystals.

OBJECTIVE: To characterize chondrocyte alkaline phosphatase (ALP) distribution and expression in cartilage and monolayer cultures. METHODS: Sections of bovine articular cartilage or chondrocyte monolayer cultures were stained for ALP activity. Surface ALP was released with bacterial phosphatidylinositol specific phospholipase C (PI-PLC). The levels of ALP mRNA were determined by Northern blot analysis using a cDNA probe to bovine ALP. RESULTS: Chondrocyte ALP activity dissolves calcium pyrophosphate dihydrate (CPPD) crystals. About 5% of the total chondrocytes contained ALP activity. The ALP positive cells were present only in the deep layers of articular cartilage adjacent to the subchondral bone. Thirty to forty percent of the total ALP activity was present on the chondrocyte surface and was released by PI-PLC. Both chondrocyte ALP activity and mRNA levels decreased with time in culture. However, continuous dexamethasone treatment stimulated the expression of ALP in ALP positive chondrocytes, sufficiently to replace all of the chondrocyte surface ALP released following PI-PLC treatment. CONCLUSION: Since ALP hydrolyzes pyrophosphate and dissolves CPPD crystals, our data suggest that regulation of chondrocyte ALP activity and expression in cartilage may prove useful for the development of a specific therapy CPPD arthropathy.

Alkaline Phosphatase↗
PubMed1993

Lumbar spinal stenosis secondary to calcium pyrophosphate crystal deposition (pseudogout).

A 62-year-old man demonstrated symptoms, signs, and radiographic evidence of lumbar spinal stenosis and intraoperative pathologic findings of tophaceous deposition in the ligamentum flavum. Although there have been reports of cervical calcium pyrophosphate dihydrate crystal deposition (CPPD) with neurologic compression, this report appears to be the first case of lumbar spinal stenosis secondary to CPPD. Cervical calcium pyrophosphate dihydrate crystal deposition should be added to the differential diagnosis of spinal stenosis.

Arthrodesis↗
PubMed1996

Inorganic pyrophosphate generation from adenosine triphosphate by cell-free human synovial fluid.

OBJECTIVE: To quantify inorganic pyrophosphate (PPi) 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] 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. 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 < 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. CONCLUSION: 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.

Adenosine Triphosphate↗
PubMed1996

Mixed monosodium urate and calcium pyrophosphate crystal-induced arthropathy. A review of seventeen cases.

Among 4620 synovial fluid specimens examined in our laboratory from 1989 through 1992, 18.3% contained crystals (calcium pyrophosphate dihydrate, 55.7%; monosodium urate, 42%). Both calcium pyrophosphate dihydrate and monosodium urate crystals were found in 17 patients, usually in specimens obtained from the knee. Only five of these 17 patients had a known history of gout. Among the patients with a mixed crystal composition, the females had a higher mean age (78.5 years) than the males (53.2 years), in keeping with the fact that gout is exceedingly rare in premenopausal women. The two youngest men (40 and 45 years) had a history of meniscectomy, which is a known risk factor for early chondrocalcinosis.

Adult↗
PubMed1999

Derivatives of 1,4-dihydropyridines as modulators of ascorbate-induced lipid peroxidation and high-amplitude swelling of mitochondria, caused by ascorbate, sodium linoleate and sodium pyrophosphate.

A group of 26 2,6-dimethyl-3,5-disubstituted and 2,6-dimethyl-3,4, 5-trisubstituted-1,4-dihydropyridines (1,4-H(2)Py=1,4-DHPs) and five related pyridines were studied as inhibitors of rat liver mitochondrial swelling and O(2) uptake by ascorbic acid-dependent lipid peroxidation (LP) and as modulators of mitochondrial swelling induced by Na(+)-linoleate or Na(+)-pyrophosphate. 1,4-DHPs studied include 4-unsubstituted and 4-methyl- and 4-phenyl-substituted 3, 5-dialkoxycarbonylderivatives of 2,6-dimethyl-1,4-DHP with variations in alkoxy chain length and composition, 4-unsubstituted and 4-methyl-, 4-aryl- and 4-pyridyl-substituted 3, 5-dianilidocarbonylderivatives, and a structurally related group of 3,5-dipyridylamidocarbonylderivatives. Many 1,4-DHPs possess marked antioxidant (AO) and membrane stabilizing activity, expressed as the mitochondrial swelling (deltaA(520)/t) and/or O(2) uptake rate decrease (V(0)/V) as well as prolongation of the induction period (tau/tau(0)) of mitochondrial swelling and/or O(2) uptake at ascorbic acid-dependent LP of rat liver mitochondria. 4-Unsubstituted 3,5-dialkoxycarbonyl-2,6-dimethyl-1,4-DHPs, as well as 4-unsubstituted or those possessing lipophylic 4-aryl- groups 3, 5-diamido-2,6-dimethyl-1,4-DHPs, reveal marked AO and membrane stabilizing properties. Oxidized (heteroaromatized) derivatives have minimal activity. Perhaps 1,4-DHPs preferably act as antioxidants on stages of initiation and prolongation of LP chain reactions at low concentrations: IC(50) (when V(0)/V or tau/tau(0)=2) are 0.1 microM to 100 microM. At 100 microM 3,5-di-p-hydroxyphenoxycarbonyl- and 3, 5-di-p-tolyloxycarbonyl-2,6-dimethyl-1,4-DHPs, as well as 3, 5-diethoxycarbonyl-2,6-dimethylpyridine (oxidized form of Hantzsch ester) and 3,5-diamyloxycarbonyl-2,6-dimethylpyridine, alter the mitochondrial swelling rate in the presence of natural protonophore Na(+)-linoleate (0.063 mM and 0.125 mM). 3,5-Di-n-butyloxycarbonyl-2, 6-dimethyl-1,4-DHP at 100 microM completely stops mitochondrial swelling in the presence of 0.8 mM Na(+)-pyrophosphate. In the presence of many of the 1,4-DHPs, the lipid peroxidation process was inhibited. However, the swelling process could be prolonged, promoted, accelerated or inhibited-depending on 1,4-DHPs structure, concentration, the type of initiators of the swelling process and the medium composition.

Animals↗
PubMedDate not supplied

Roentgenographic aspects of calcium pyrophosphate dihydrate crystal deposition disease (pseudogout).

A comprehensive review of the roentgenographic features of calcium pyrophosphate crystal deposition disease (pseudogout) is presented. The roentgenographic techniques are reviewed. Those recommended as optimal are as follows: for small peripheral joints, industrial Type M film with contact exposure; for large central joints, direct radiographic magnification (3-4 X) with a microfocus tube. The classic appearances of articular chondrocalcinosis in fibrocartilage, hyaline cartilage, and capsular structures are discussed. Distinctive features of pyrophosphate arthropathy, including the distribution of involvement and the degree of destruction, are emphasized. Finally, the relationship among articular chondrocalcinosis, arthropathy, microscopic crystals, and symptoms is discussed.

Adult↗
PubMed2000

Expression of cartilage intermediate layer protein/nucleotide pyrophosphohydrolase parallels the production of extracellular inorganic pyrophosphate in response to growth factors and with aging.

OBJECTIVE: To evaluate the role of the extracellular inorganic pyrophosphate (ePPi)-generating ectoenzyme cartilage intermediate layer protein/nucleotide pyrophosphohydrolase (CILP/NTPPH) in chondrocyte PPi elaboration, we studied CILP/NTPPH expression in response to growth factors during aging. METHODS: Porcine chondrocytes from adult (3-4-year-old) and young (2-week-old) animals were stimulated with transforming growth factor beta1 (TGFbeta1), which enhances ePPi elaboration, and/or insulin-like growth factor 1 (IGF-1), which diminishes ePPi elaboration. Measurements of ePPi, NTPPH enzyme activity, Western blot analysis, reverse transcriptase-polymerase chain reaction (RT-PCR), and Northern blot analysis were performed. RESULTS: Elaboration of ePPi into conditioned media from adult chondrocytes was significantly increased by TGFbeta1 and significantly inhibited by IGF-1, but no significant differences were observed in young chondrocytes. The protein levels of CILP/NTPPH by Western analysis in the media from adult and young porcine chondrocytes were increased by TGFbeta1. RT-PCR and Northern analysis showed that CILP/NTPPH messenger RNA (mRNA) expression in both adult and young chondrocytes was increased by TGFbeta1 and decreased by IGF-1, but these changes were less significant in the young chondrocytes. Basal and TGFbeta1-up-regulated levels of CILP/NTPPH expression were higher in adult chondrocytes than in young chondrocytes. CONCLUSION: These results provide evidence that CILP/NTPPH expression and ePPi elaboration are concomitantly stimulated by TGFbeta1 and down-regulated by IGF-1, especially in adult chondrocytes, implicating CILP/NTPPH as a functional participant in ePPi elaboration. Increased CILP/NTPPH mRNA expression in chondrocytes derived from aged animals compared with young animals might promote the formation of calcium pyrophosphate dihydrate crystals in aged cartilage.

Aging↗
PubMed2002

Up-regulated expression of cartilage intermediate-layer protein and ANK in articular hyaline cartilage from patients with calcium pyrophosphate dihydrate crystal deposition disease.

OBJECTIVE: Excess accumulation of extracellular inorganic pyrophosphate (ePPi) in aged human cartilage is crucial in calcium pyrophosphate dihydrate (CPPD) crystal formation in cartilage matrix. Two sources of ePPi are ePPi-generating ectoenzymes (NTPPPH) and extracellular transport of intracellular PPi by ANK. This study was undertaken to evaluate the role of NTPPPH and ANK in ePPi elaboration, by investigating expression of NTPPPH enzymes (cartilage intermediate-layer protein [CILP] and plasma cell membrane glycoprotein 1 [PC-1]) and ANK in human chondrocytes from osteoarthritic (OA) articular cartilage containing CPPD crystals and without crystals. METHODS: Chondrocytes were harvested from knee cartilage at the time of arthroplasty (OA with CPPD crystals [CPPD], n = 8; OA without crystals [OA], n = 10). Normal adult human chondrocytes (n = 1) were used as a control. Chondrocytes were cultured with transforming growth factor beta1 (TGFbeta1), which stimulates ePPi elaboration, and/or insulin-like growth factor 1 (IGF-1), which inhibits ePPi elaboration. NTPPPH and ePPi were measured in the media at 48 hours. Media CILP, PC-1, and ANK were determined by dot-immunoblot analysis. Chondrocyte messenger RNA (mRNA) was extracted for reverse transcriptase-polymerase chain reaction to study expression of mRNA for CILP, PC-1, and ANK. NTPPPH and ANK mRNA and protein were also studied in fresh frozen cartilage. RESULTS: Basal ePPi elaboration and NTPPPH activity in conditioned media from CPPD chondrocytes were elevated compared with normal chondrocytes, and tended to be higher compared with OA chondrocytes. Basal expression of mRNA for CILP (chondrocytes) and ANK (cartilage) was higher in both CPPD chondrocytes and CPPD cartilage extract than in OA or normal samples. PC-1 mRNA was less abundant in CPPD chondrocytes and cartilage extract than in OA chondrocytes and extract, although the difference was not significant. CILP, PC-1, and ANK protein levels were similar in CPPD, OA, and normal chondrocytes or cartilage extracts. Both CILP and ANK mRNA expression and ePPi elaboration were stimulated by TGFbeta1 and inhibited by IGF-1 in chondrocytes from all sources. CONCLUSION: CILP and ANK mRNA expression correlates with chondrocyte ePPi accumulation around CPPD and OA chondrocytes, and all respond similarly to growth factor stimulation. These findings suggest that up-regulated CILP and ANK expression contributes to higher ePPi accumulation from CPPD crystal-forming cartilage.

Adult↗
PubMed1988

Synovial fluid inorganic pyrophosphate concentration and nucleotide pyrophosphohydrolase activity in basic calcium phosphate deposition arthropathy and Milwaukee shoulder syndrome.

Synovial fluid (SF) inorganic pyrophosphate (PPi) concentration is elevated in calcium pyrophosphate dihydrate (CPPD) crystal deposition arthropathy. Since CPPD and basic calcium phosphate (BCP) crystals often are present in the same joints, we determined [PPi] and activity of the PPi-generating enzyme, nucleotide pyrophosphohydrolase (NPPH), in SF from the joints of patients with various arthropathies, including those with BCP crystals. We found elevated SF [PPi] in joints with BCP crystals, as well as in joints with CPPD crystals. The presence of BCP crystals in synovial fluids was also predictive of elevated NPPH activity.

Calcium Phosphates↗
PubMed1990

Cartilage inorganic pyrophosphate elaboration is independent of sulfated glycosaminoglycan synthesis.

Inorganic pyrophosphate (PPi), a product of glycosaminoglycan synthesis, may be cosecreted with matrix proteoglycan to reach the extracellular site where calcium pyrophosphate dihydrate crystals form. To test this hypothesis, sulfated glycosaminoglycan synthesis by articular cartilage in culture was stimulated or inhibited while the effect on extracellular PPi was measured. When stimulated by 0.8 mM xyloside to increase 35SO4 incorporation (mean +/- SEM % of control 183 +/- 16, n = 5), PPi accumulation changed little (from 54 +/- 6 pmoles/mg to 63 +/- 8 pmoles/mg of cartilage wet weight). Inhibition of sulfation with monensin or diethylcarbamazine disproportionately lowered 35SO4 incorporation compared with PPi elaboration. Using 60 mM diethylcarbamazine, PPi production was preserved (105 +/- 8% mean +/- SEM) compared with control cultures, while sulfation was markedly inhibited (7 +/- 1%). This dissociation of sulfate incorporation and PPi secretion indicates that it is not likely that glycosaminoglycan sulfation is the source of the PPi that escapes from chondrocytes to participate in the formation of extracellular crystals.

Animals↗
PubMed1991

Transforming growth factor beta 1 stimulates inorganic pyrophosphate elaboration by porcine cartilage.

The overproduction of inorganic pyrophosphate (PPi) by cartilage is thought to be a key element in the formation of calcium pyrophosphate dihydrate (CPPD) crystals in joints, and the subsequent development of pseudogout or chondrocalcinosis. We report herein that transforming growth factor beta 1 (TGF beta 1), alone and in synergy with epidermal growth factor (EGF) or TGF alpha, markedly stimulates PPi elaboration by porcine articular cartilage in organ culture and monolayer culture. This effect is not seen with platelet-derived growth factor, basic fibroblast growth factor, or insulin-like growth factor types 1 and 2, substances which also affect chondrocyte metabolism or are mitogenic. TGF beta 1 produces only a modest increase in nucleoside triphosphate pyrophosphohydrolase (NTPPPH), a chondrocyte ectoenzyme that produces PPi; this implies the existence of other pathways for PPi elaboration. TGF beta 1 is present in joint fluid and cartilage. TGF beta 1, TGF alpha, and EGF are the first known physiologic modifiers of cartilage PPi production. They provide a novel model for the study of CPPD crystal formation in cartilage, as well as new insights into the pathogenesis of this common affliction of aging.

Animals↗
PubMed1979

Reliability and reproducibility of interpretation of 99mtechnetium pyrophosphate myocardial scintigrams.

The interpretations of 156 99mtechnetium pyrophosphate myocardial scintigrams by four observers were analyzed in order to determine the reliability and reproducibility of the subjective process of reading scintigrams. The scintigrams were scored on an integral scale from 0 to 4, depending upon the degree of myocardial radionuclide accumulation, and the site and nature of uptake were specified. Exact agreement upon score was generally poor but approximate concurrence of interpretation was good (90.4 and 92.5% inter- and intra-observer agreement, respectively). There was somewhat less agreement on scintigrams with the higher scores of 3 and 4 (83.3 and 78.0%, respectively). A high level of concurrence upon the differentiation between diffuse and localized uptake, and upon the site of uptake, was found. We conclude that only approximate rather than exact agreement of individual readers' interpretations can be expected in this subjective technique, that scintigrams with higher degrees of radionuclide accumulation produce slightly greater observer disagreement, and that variability of interpretation could account for some of the diagnostic inaccuracy of 99mtechnetium pyrophosphate myocardial scintigraphy.

Angina Pectoris↗
PubMed1996

Identification of isoprenyl modified proteins metabolically labeled with [3H]farnesyl- and [3H]geranylgeranyl-pyrophosphate.

Here we describe a direct approach for two-dimensional (2-D) gel mapping of proteins that are modified by post-translational isoprenylation in mammalian cells. Briefly, transformed human amnion cells (AMA) and transfected COS-1 cells were metabolically labeled with either [3H]farnesyl-pyrophosphate or [3H]geranylgeranyl-pyrophosphate following treatment with lovastatin, which blocks the synthesis of mevalonic acid. The proteins were then separated by 2-D gel electrophoresis and electrotransferred to nitrocellulose filters. The membranes were immersed in dimethyl ether, containing 10% of 2,5-diphenyloxazole prior to fluorography. Over 40 [3H]farnesyl-labeled proteins and over 25 [3H]geranylgeranylated proteins were identified on the 2-D autoradiograms. Several [3H]farnesyl-labeled proteins exhibited the same coordinates (M(r) and pI) as their [3H]geranylgeranylated counterparts, raising the possibility that they may be substrates for both farnesyl and geranylgeranyl transferase(s). The approach offers high resolution of both farnesylated and geranylgeranylated proteins and it may serve as a powerful tool for the identification of hitherto unknown prenylated proteins as well as for the determination of prenylated protein levels, type of isoprenoid modification, and possible changes in protein prenyltransferase activity.

Amnion↗
PubMed1993

Solid phase DNA minisequencing by an enzymatic luminometric inorganic pyrophosphate detection assay.

A solid phase DNA sequencing method for non-radioactive detection of single base changes without the need for electrophoresis is presented. The concept relies on the detection of DNA polymerase activity by an enzymatic luminometric inorganic pyrophosphate detection assay (P. Nyrén, 1987, Anal. Biochem. 167, 235-238). Immobilized DNA fragments amplified with the polymerase chain reaction are used as template. A detection primer is annealed in front of the mutation and four aliquots of this mixture are incubated with DNA polymerase and one of the four different dideoxynucleotides. The presence or absence of an incorporated dideoxynucleotide is thereafter monitored by the release of inorganic pyrophosphate during the following primer extension step. We show that the concept can be used for sequencing of single bases as well as stepwise analysis of several subsequent bases.

Base Sequence↗
PubMed1993

Detection of glycinamide ribonucleotide by HPLC with pulsed amperometry: application to the assay for glutamine: 5-phosphoribosyl-1-pyrophosphate amidotransferase (EC 2.4.2.14).

An assay method is described for measuring the catalytic activity of glutamine: 5-phosphoribosyl-1-pyrophosphate amidotransferase (EC 2.4.2.14), the first enzyme in the de novo purine biosynthetic pathway. Phosphoribosylamine, the unstable product of the enzymatic reaction, is trapped by glycinamide ribonucleotide synthetase (EC 6.3.4.13), the second enzyme in the pathway, to form the stable product glycinamide ribonucleotide. Glycinamide ribonucleotide is resolved by ion-exchange HPLC at alkaline pH and quantified by the use of a pulsed amperometric detector. The assay is specific, reproducible, rapid (15 min chromatography, including column recycling and stabilization), convenient (no radioactive materials used), linear at a wide range of glycinamide ribonucleotide concentrations, and sensitive at subnanomole levels even with crude extracts. Both the glutamine- and ammonia-dependent activity of glutamine: 5-phosphoribosyl-1-pyrophosphate amidotransferase can be accurately quantified.

Amidophosphoribosyltransferase↗
PubMed2001

Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation.

The loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that uses only one type of enzyme. One of the characteristics of the LAMP method is its ability to synthesize extremely large amount of DNA. Accordingly, a large amount of by-product, pyrophosphate ion, is produced, yielding white precipitate of magnesium pyrophosphate in the reaction mixture. Judging the presence or absence of this white precipitate allows easy distinction of whether nucleic acid was amplified by the LAMP method. Since an increase in the turbidity of the reaction mixture according to the production of precipitate correlates with the amount of DNA synthesized, real-time monitoring of the LAMP reaction was achieved by real-time measurement of turbidity.

Base Sequence↗