Diagnosis of calcium pyrophosphate dihydrate deposition disease by fine needle aspiration biopsy: a case report

BACKGROUND: Calcium pyrophosphate dihydrate deposition disease is a relatively rare disease with variable clinical presentations. CASE: A 73-year-old man presented with worsening lower back pain and fever. Fine needle aspiration biopsy of the lumbar vertebral bodies (L3-L4) revealed abundant neutrophils admixed with small, birefringent, rhomboid crystals in Diff-Quik-stained smears. These crystals were confirmed as calcium pyrophosphate dihydrate on cell block sections. A diagnosis of osteomyelitis and calcium pyrophosphate dihydrate deposition disease was rendered. The patient was treated with antibiotics and responded well. CONCLUSION: Calcium pyrophosphate dihydrate deposition disease can be diagnosed by fine needle aspiration biopsy, and an accurate diagnosis can be greatly facilitated by cell block sections. However, such a diagnosis may be neglected if the specimen is not carefully inspected.     

Autosomal dominant familial calcium pyrophosphate dihydrate deposition disease is caused by mutation in the transmembrane protein ANKH

Familial autosomal dominant calcium pyrophosphate dihydrate (CPPD) chondrocalcinosis has previously been mapped to chromosome 5p15. We have identified a mutation in the ANKH gene that segregates with the disease in a family with this condition. ANKH encodes a putative transmembrane inorganic pyrophosphate (PPi) transport channel. We postulate that loss of function of ANKH causes elevated extracellular PPi levels, predisposing to CPPD crystal deposition.     

Refinement of the chromosome 5p locus for familial calcium pyrophosphate dihydrate deposition disease.

Familial calcium pyrophosphate dihydrate deposition disease (CPPDD) is a disease of articular cartilage that is radiographically characterized by chondrocalcinosis due to the deposition of calcium-containing crystals in affected joints. We have documented the disease in an Argentinean kindred of northern Italian ancestry and in a French kindred from the Alsace region. Both families presented with a common phenotype including early age at onset and deposition of crystals of calcium pyrophosphate dihydrate in a similar pattern of affected joints. Affected family members were karyotypically normal. Linkage to the short arm of chromosome 5 was observed, consistent with a previous report of linkage of the CPPDD phenotype in a large British kindred to the 5p15 region. However, recombinants in the Argentinean kindred have enabled us to designate a region<1 cM in length between the markers D5S416 and D5S2114 as the CPPDD locus.     

Monosodium urate and calcium pyrophosphate crystals differentially activate the excitation-response coupling sequence of human neutrophils

The activation patterns of human neutrophils elicited by unopsonized monosodium urate and calcium pyrophosphate dihydrate crystals were investigated. The parameters chosen, the mobilization of calcium and the synthesis of leukotrienes, are generally accepted to be relevant to the activation of the cells and their pathophysiological roles. Both particles were found to elicit increases in cytoplasmic free calcium and leukotriene synthesis. However, the rank order of potency of these two stimuli was found to be sharply dependent on the test chosen. Monosodium urate crystals were significantly more effective than calcium pyrophosphate dihydrate crystals in terms of calcium mobilization, while the latter are more potent at inducing leukotriene synthesis. These results demonstrate that these two phagocytic particles which are related to separate inflammatory joint diseases differentially activate the excitation-response coupling sequence of human neutrophils.     

P5L mutation in Ank results in an increase in extracellular inorganic pyrophosphate during proliferation and nonmineralizing hypertrophy in stably transduced ATDC5 cells

Ank is a multipass transmembrane protein that regulates the cellular transport of inorganic pyrophosphate. In the progressive ankylosis (ank) mouse, a premature termination mutation at glutamic acid 440 results in a phenotype characterized by inappropriate deposition of basic calcium phosphate crystals in skeletal tissues. Mutations in the amino terminus of ANKH, the human homolog of Ank, result in familial calcium pyrophosphate dihydrate deposition disease. It has been hypothesized that these mutations result in a gain-of-function with respect to the elaboration of extracellular inorganic pyrophosphate. To explore this issue in a mineralization-competent system, we stably transduced ATDC5 cells with wild-type Ank as well as with familial chondrocalcinosis-causing Ank mutations. We evaluated the elaboration of inorganic pyrophosphate, the activity of pyrophosphate-modulating enzymes, and the mineralization in the transduced cells. Expression of transduced protein was confirmed by quantitative real-time PCR and by ELISA. Levels of inorganic pyrophosphate were measured, as were the activities of nucleotide pyrophosphatase phosphodiesterase and alkaline phosphatase. We also evaluated the expression of markers of chondrocyte maturation and the nature of the mineralization phase elaborated by transduced cells. The cell line expressing the proline to leucine mutation at position 5 (P5L) consistently displayed higher levels of extracellular inorganic pyrophosphate and higher phosphodiesterase activity than the other transduced lines. During hypertrophy, however, extracellular inorganic pyrophosphate levels were modulated by alkaline phosphatase activity in this cell system, resulting in the deposition of basic calcium phosphate crystals only in all transduced cell lines. Cells overexpressing wild-type Ank displayed a higher level of expression of type X collagen than cells transduced with mutant Ank. Other markers of hypertrophy and terminal differentiation, such as alkaline phosphatase, osteopontin, and runx2, were not significantly different in cells expressing wild-type or mutant Ank in comparison with cells transduced with an empty vector or with untransduced cells. These results suggest that the P5L Ank mutant is capable of demonstrating a gain-of-function with respect to extracellular inorganic pyrophosphate elaboration, but this effect is modified by high levels of expression of alkaline phosphatase in ATDC5 cells during hypertrophy and terminal differentiation, resulting in the deposition of basic calcium phosphate crystals.     

Stimulation of inorganic pyrophosphate elaboration by cultured cartilage and chondrocytes

Inorganic pyrophosphate elaboration by articular cartilage may favor calcium pyrophosphate dihydrate crystal deposition. Frequently crystal deposits form in persons affected with metabolic diseases. The cartilage organ culture system was used to model these metabolic conditions while measuring the influence on extracellular pyrophosphate elaboration. Alterations of ambient pH, thyroid stimulating hormone levels, and parathyroid hormone levels did not change pyrophosphate accumulation in the media. However, subphysiologic ambient calcium concentrations (25, 100, 500 microM) increased pyrophosphate accumulation about chondrocytes 3- to 10-fold. Low calcium also induced release of [14C]adenine-labeled nucleotides from chondrocytes, potential substrates for generation of extracellular pyrophosphate by ectoenzymes. Exposing cartilage to 10% fetal bovine serum also enhanced by 50% the egress of inorganic pyrophosphate from the tissue.     

P5L mutation in Ank results in an increase in extracellular inorganic pyrophosphate during proliferation and nonmineralizing hypertrophy in stably transduced ATDC5 cells

Ank is a multipass transmembrane protein that regulates the cellular transport of inorganic pyrophosphate. In the progressive ankylosis (ank) mouse, a premature termination mutation at glutamic acid 440 results in a phenotype characterized by inappropriate deposition of basic calcium phosphate crystals in skeletal tissues. Mutations in the amino terminus of ANKH, the human homolog of Ank, result in familial calcium pyrophosphate dihydrate deposition disease. It has been hypothesized that these mutations result in a gain-of-function with respect to the elaboration of extracellular inorganic pyrophosphate. To explore this issue in a mineralization-competent system, we stably transduced ATDC5 cells with wild-type Ank as well as with familial chondrocalcinosis-causing Ank mutations. We evaluated the elaboration of inorganic pyrophosphate, the activity of pyrophosphate-modulating enzymes, and the mineralization in the transduced cells. Expression of transduced protein was confirmed by quantitative real-time PCR and by ELISA. Levels of inorganic pyrophosphate were measured, as were the activities of nucleotide pyrophosphatase phosphodiesterase and alkaline phosphatase. We also evaluated the expression of markers of chondrocyte maturation and the nature of the mineralization phase elaborated by transduced cells. The cell line expressing the proline to leucine mutation at position 5 (P5L) consistently displayed higher levels of extracellular inorganic pyrophosphate and higher phosphodiesterase activity than the other transduced lines. During hypertrophy, however, extracellular inorganic pyrophosphate levels were modulated by alkaline phosphatase activity in this cell system, resulting in the deposition of basic calcium phosphate crystals only in all transduced cell lines. Cells overexpressing wild-type Ank displayed a higher level of expression of type X collagen than cells transduced with mutant Ank. Other markers of hypertrophy and terminal differentiation, such as alkaline phosphatase, osteopontin, and runx2, were not significantly different in cells expressing wild-type or mutant Ank in comparison with cells transduced with an empty vector or with untransduced cells. These results suggest that the P5L Ank mutant is capable of demonstrating a gain-of-function with respect to extracellular inorganic pyrophosphate elaboration, but this effect is modified by high levels of expression of alkaline phosphatase in ATDC5 cells during hypertrophy and terminal differentiation, resulting in the deposition of basic calcium phosphate crystals.     

Identification of adenine-N9-(methoxy)ethyl-β-bisphosphonate as NPP1 inhibitor attenuates NPPase activity in human osteoarthritic chondrocytes

Purinergic Signalling - Overproduction of extracellular diphosphate due to hydrolysis of ATP by NPP1 leads to pathological calcium diphosphate (pyrophosphate)&nbsp;dihydrate deposition (CPPD)...     

Clinical and roentgenographic aspects of pseudogout: a study of 50 cases and a review.

Pseudogout, defined as recurrent acute arthritis due to intrasynovial deposition of calcium pyrophosphate dihydrate crystals, is a relatively common arthritic disorder of the elderly. The clinical and roentgenographic aspects of 50 cases of pseudogout in hospitalized patients are reviewed in this paper. Oligoarticular and polyarticular episodes were observed in half of these patients. Antecedent problems included infection, trauma, surgery and vascular events. Consistent with previous reports, most patients had roentgenographic evidence of chondrocalcinosis. A third had asymptomatic capsular or periarticular calcific deposits or both, and a third had pyrophosphate arthropathy, a progressive, destructive, accelerated form of osteoarthritis. An attack of pseudogout may offer a clue to the presence of an unsuspected metabolic disease, such as primary hyperparathyroidism or idiopathic hemochromatosis.     

Inflammatory microcrystals induce murine macrophage survival and DNA synthesis

The interaction of particulates with resident macrophages is a consistent feature in certain forms of crystal-induced inflammation, for example, in synovial tissues, lung, and the peritoneum. The mitogenic activity of basic calcium phosphate (BCP) crystals and calcium pyrophosphate dihydrate (CPPD) crystals on synovial fibroblasts has been considered relevant to the synovial hyperplasia observed in crystal-induced arthritis. The aim of the study was to determine whether microcrystals such as these could enhance macrophage survival and induce DNA synthesis, thus indicating that they may contribute to the tissue hyperplasia.     

Permanent Stained Preparations of Synovial Fluid for Detection of Calcium Compounds Using Alizarin Red S

Permanent preparations of air dried synovial fluids were prepared by staining calcium compounds with alizarin red S stain; each slide was coverslipped with Permount. Variables studied were: (a) concentration of the solution of alizarin red S, (b) pH of staining solution, (c) time of incubation in staining solution and aqueous and ethanolic content of staining solution. The staining effect of each solution was tested on calcium pyrophosphate dihydrate, calcium oxalate, apatite and monosodium urate (MSU). Of all the solutions, best results were obtained with 0.25% alizarin red S in 50% ethanol at pH 7.0 for 30 min. With this solution, the calcium-containing compounds were well stained. MSU did not stain and still preserved negative birefringence on polarizaton. Fixation of smears with ethanol served a double purpose: It fixed the slides without dissolving or removing MSU or the calcium compounds, yet it did dissolve five corticosteroids commonly used for intra-articular injection which may interfere with interpretation of compensated polarized light microscopy of synovial fluids.     

Two cases of acute pseudogout attack following parathyroidectomy.

Two cases of acute attack of pseudogout associated with primary hyperparathyroidism are reported. Case 1 suffered from acute pain and swelling of the right ankle and dorsal of the right foot. Case 2 suffered from unknown fever and pain of the bilateral jaw, shoulder, elbow, wrist and knee joints. Postoperative radiological studies revealed the association of chondrocalcinosis in both cases. Synovial fluid in case 2 was aspirated and analyzed for calcium pyrophosphate dihydrate crystal by microscopic examination.     

Evidence that ecto-nucleoside-triphosphate pyrophosphatase serves in the generation of extracellular inorganic pyrophosphate in human bone and articular cartilage

Extracellular inorganic pyrophosphate (PPi) is important in the regulation of mineralisation of bone, and in the pathogenesis of chondrocalcinosis, an arthritic disease in which calcium pyrophosphate dihydrate crystals form in articular cartilage. Nucleoside-triphosphate pyrophosphatase, which catalyses the formation of PPi, was previously observed at the surface of human articular chondrocytes in culture. A similar enzyme has been identified in osteoblast-like human bone cells in culture, and is active towards purine and pyrimidine nucleoside triphosphates. The enzyme has high affinity for ATP and is located on the cell surface, and thus could serve in the generation of extracellular PPi. Moreover, no other mechanism for the catabolism of small amounts of exogenous ATP is present in human bone cells. Further evidence for ecto-nucleoside-triphosphate pyrophosphatase serving in the generation of extracellular PPi in articular cartilage and bone was obtained by studying the ability of alternative substrates (which do not yield PPi) to inhibit generation of PPi from ATP. In both articular chondrocytes and bone cells, the enzyme exhibited an apparent preference for ATP over dinucleotide and phosphodiester substrates. Some potential inhibitors of the enzyme activity were also studied in both cell types. ADP moderately inhibited the activity but two bisphosphonate drugs were only slightly inhibitory.     

Prevalence of chondrocalcinosis in patients with primary hyperparathyroidism in Japan.

One hundred and thirty-two consecutive patients with primary hyperparathyroidism were studied preoperatively for the presence of chondrocalcinosis, the roentgenographic marker of calcium pyrophosphate dihydrate (CPPD) crystal deposition disease, by obtaining radiographs of knees, wrists and pelvis. Chondrocalcinosis was found in 8 patients (6.1%), each of whom was over 50 years of age. In 72 of the patients over 50 years of age, the prevalence of chondrocalcinosis in the hyperparathyroid patients (11.1%) was greater than that found in 72 control patients (2.8%) with thyroid nodular disease who were exactly matched for age and sex, but the difference was not significant. The prevalence of chondrocalcinosis in the hyperparathyroid patients sharply increased with age. In the group in their 50's it was 4.4%, rising to 15.8% in patients in their 60's and reaching 37.5% for those over 70 years of age. Patients with chondrocalcinosis were significantly older than those without this finding (p < 0.005). Those with chondrocalcinosis also had significantly higher preoperative serum calcium levels than those without it (p < 0.05). While chondrocalcinosis was detected by taking joint radiographs in all patients with primary hyperparathyroidism, acute arthritis (pseudogout attack) occurred in only 2 of the 132 patients (1.5%) after parathyroidectomy, but this represents 25% (2 of 8) of those who had chondrocalcinosis. An attack of pseudogout may therefore be one of the most common postoperative complications of parathyroid surgery in the elderly. Considering the low incidence of pseudogout attack following parathyroidectomy, preoperative radiological studies of the knee joints are sufficient to screen for chondrocalcinosis and are recommended for patients over 60 years old in Japan.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular dynamics simulation studies of the effect of phosphocitrate on crystal-induced membranolysis

In this study, following our earlier work on calcium pyrophosphate dihydrate (CPPD) crystal-induced membranolysis, we demonstrate, using the CHARMM method of molecular dynamics simulation, the protective role of phosphocitrate (PC) against solvated dimyristoyl phosphatidylcholine phospholipid bilayer disintegration on contact with the CPPD crystal. Our molecular dynamics simulations studies show that coverage of the CPPD crystal with a layer of phosphocitrate molecules results in the conservation of phospholipid bilayer integrity. We show that the rupture of the lipid bilayer in presence of CPPD and the protective effect of PC are primarily due to electrostatic interactions. The protective role of PC, which may also play an important and potentially therapeutic function against crystal-induced membranolysis is also discussed.     

Phagocytosis of hydroxyapatite or calcium pyrophosphate dihydrate crystals by rabbit articular chondrocytes stimulates release of collagenase, neutral protease, and prostaglandins E2 and F2 alpha

Synthetic hydroxyapatite (HA) and calcium pyrophosphate dihydrate (CPPD) microcrystals are phagocytosed by rabbit articular-cartilage chondrocytes in primary culture. The ingestion of crystals greatly stimulated the release of collagenase, neutral protease, and prostaglandins E2 and F2 alpha into the ambient medium. Lactate dehydrogenase was not released by either crystal despite electron microscopic evidence of cell damage by HA crystals (partial loss of phagolysosomal membrane and increased myelin figures). HA, but not CPPD crystals, stimulated release of beta-glucuronidase. HA crystal concentrations from 50 to 200 micrograms ml-1 induced a dose-dependent release of collagenase and of extracellular protein. Both phagocytosis and collagenase release were greatly attenuated when HA crystals were added to the chondrocyte monolayers in the absence of serum. As HA and CPPD crystals have been identified in human articular cartilage in association with degenerative changes, it is possible that the cell-crystal interaction described here may be pathogenetically important.     

Promotion of neutrophil adherence to human osteoblasts by microcrystals and f-Met-Leu-Phe

Human osteoblast-like cells (hOB) stimulated by monosodium urate monohydrate (MSUM) or calcium pyrophosphate dihydrate (CPPD) microcrystals produce the neutrophil chemoattractant IL-8. We investigated whether human neutrophils can adhere to hOB and respond to hOB preactivated by MSUM, CPPD, or by f-Met-Leu-Phe (fMLP). Confluent hOB were coincubated with human blood neutrophils in the presence of MSUM, CPPD or fMLP. MSUM, CPPD, and fMLP stimulated a significant adherence of neutrophils to hOB after a 1h incubation. This effect was not abrogated by pretreating the cells with an anti-CD18 mAb. MSUM stimulated more efficiently the adherence of neutrophils to non-preactivated hOB while CPPD were more efficient when hOB were preactivated. Crystal-free conditioned media from MSUM- or CPPD-stimulated hOB mobilized intracellular free calcium in human neutrophils. Thus, microcrystals were powerful promoters of neutrophil adherence to hOB via a CD18-independent mechanism. The bacterial peptide fMLP also stimulated the adherence of neutrophils to hOB. Functional neutrophil-hOB interactions could be important in bone pathophysiology of crystal- or infection-associated arthritis.     

Phosphate Solubility and the Cyanate-Mediated Synthesis of Pyrophosphate

The justification for a less alkaline primordial ocean (than present) is briefly reviewed, along with constraints on aqueous phosphate under such conditions. Based on the assumption that CaHPO₄ dihydrate determined the availability of phosphorus species, we have carried out laboratory simulations to determine equilibrium concentrations as a function of pH (in PIPES buffer) with added NaCl and CaCl₂. Consistent with expectations, solubility declines with higher pH and [CaCl₂], but increases only slightly with [NaCl]. Significantly, PIPES shows no specific effect on the dissolution beyond its influence on pH and ionic strength. Data are also presented on the synthesis of pyrophosphate from the NaOCN/CaHPO₄·2H₂O system, which could have provided a source of this phosphate anhydride on the early Earth.     

Biocompatible DCPD Coating Formed on AZ91D Magnesium Alloy by Chemical Deposition and Its Corrosion Behaviors in SBF

Bioactive calcium phosphate coatings were prepared on AZ91D magnesium alloy in phosphating solution in order to im- prove the corrosion resistance of the magnesium alloy in Simulated Body Fluid （SBF）. The surface morphologies and compo- sitions of the calcium phosphate coatings deposited in the phosphating bath with different compositions were investigated by Scanning Electron Microscopy （SEM） with Energy Dispersive Spectrometer （EDS） and X-ray Diffraction （XRD）. Results showed that the calcium phosphate coating was mainly composed of dicalcium phosphate dihydrate （CaHPO4o2H20, DCPD）, with Ca/P ratio of approximately 1 ： 1. The corrosion resistance was evaluated by acid drop, electrochemical polarization, elec- trochemical impedance spectroscopy and immersion tests. The dense and uniform calcium phosphate coating obtained from the optimal phosphating bath can greatly decrease the corrosion rate and hydrogen evolution rate of AZ91D magnesium alloy in SBE