Inorganic pyrophosphate metabolism in arthritis.

Once thought of as a biosynthetic waste product, over the last 2 decades PPi has become understood as an entity with a variety of biologic roles (see Table 1). Documented roles include participation in intracellular Ca++ traffic, mediation of nucleotide and iron transport, storage of molecules in cellular granules, modification of enzyme function, and modulation of mineralization. Much has been established regarding plasma, urine, and synovial fluid levels (see Fig. 1) and urinary excretion in health and disease. Derangements in intracellular PPi content of skin fibroblasts have been noted in patients with CPPD deposition arthropathy (see Table 2). Mechanisms by which elevated PPi concentration develops in synovial fluid from joints with CPPD deposition and related arthropathies have come under scrutiny. The chondrocyte is now recognized as the probable cellular source of intra-articular extracellular PPi (see Figs. 3 and 4). Special attention has been focused on two basic pathways by which chondrocytes could generate extracellular PPi (see Fig. 2). In the first mechanism, chondrocytes demonstrate a set of ectoenzymes which could work in concert to directly produce extracellular PPi. The second pathway involves the major reactions by which PPi is formed within the cell and how intracellular PPi thus formed could be transported into the extracellular space. Much future research is needed regarding these two pathways and their relative importance in the pathogenesis of CPPD crystal deposition and related arthropathies.

Inorganic pyrophosphate synthesis during methanogenesis from methylcoenzyme M by cell-free extracts of Methanobacterium thermoautotrophicum (strain delta H).

Cell-free extracts of Methanobacterium thermoautotrophicum (strain delta H) were found to contain high concentrations of inorganic pyrophosphate (up to 40 mM). The compound was accumulated by the organism despite high activity of inorganic pyrophosphatase which was found to be present in the cell extracts (1-2 mumol min-1 mg protein-1). This activity was strongly inhibited at [PPi] greater than 1.0 mM. It was demonstrated that PPi synthesis occurred during methylcoenzyme M reduction under hydrogen atmosphere: in the first stage of the reaction for each mole of methane formed one mole of PPi was produced. Inhibition of the methylcoenzyme M reduction by 2-bromoethanesulfonic acid or by high concentrations (greater than 3 microM) of tetrachlorosalicylanilide also inhibited PPi synthesis. In contrast, low concentrations (1.3 microM) of tetrachlorosalicylanilide only inhibited PPi synthesis to the same extent as the methylcoenzyme M reduction was affected. In a later stage of the methylcoenzyme M reduction, PPi synthesis dropped and a second, as yet unidentified, unstable compound was formed. Synthesis of this compound also paralleled methane formation in a stoichiometric way and was affected by the inhibiting substances in a similar way as PPi synthesis.

Guanine nucleotide and pyrophosphate activate exogenous phosphatidylinositol 4,5-bisphosphate hydrolysis in rat liver plasma membranes.

5'-guanylylimidodiphosphate (GppNHp) in the presence of deoxycholate, stimulated the phospholipase C-mediated hydrolysis of exogenous [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PIP2) to myo-[3H]inositol 1,4,5-trisphosphate in rat liver plasma membranes. Activation was not specific for guanine nucleotides as 5'-adenylylimidodiphosphate, imidodiphosphate and pyrophosphate stimulated the enzyme with similar efficacies and potencies. Enzyme activation by GppNHp was most pronounced when [3H]PIP2 was used as substrate. No added Ca++ was required for [3H]PIP2 breakdown but hydrolysis was inhibited by divalent ion chelators. GppNHp stimulation was apparent in the presence of Ca++ or Mg++ as well as chelator concentrations that partially inhibited the enzyme, indicating that this effect was not attributed to changes in affinity of these divalent cations for the enzyme or substrate. These results suggest that guanine nucleotides can stimulate the hydrolysis of exogenous [3H]PIP2 in rat liver membranes by a non-specific effect probably due to the interaction of the diphosphate moiety with the enzyme or substrate.

Can biological calcification occur in the presence of pyrophosphate?

It was shown that it is apparently impossible for any calcification to occur in a system which contains a constant, physiological concentration of inorganic pyrophosphate. These results give further support to the earlier suggestion that inhibitors of calcium phosphate crystal growth must be removed from or denied access to the site of calcification to allow for the normal formation of the mineral phase. This study also suggests that the means of assessing the relative importance of a calcification inhibitor should be altered, since it is the equilibrium concentration of an inhibitor, and not its initial concentration in an assay system, which has physiological relevance.

Calcium pyrophosphate dihydrate deposition in lumbar disc fibrocartilage.

Calcium pyrophosphate dihydrate deposits were found in the lumbar disc fibrocartilage in 4 patients undergoing surgery for spinal cord or nerve root compression. All of the patients had prior surgery at the same lumbar area. None of the patients had the articular or roentgenographic manifestations of calcium pyrophosphate deposition disease (pseudogout). Andres and Trainer have recently reported 7 similar patients. Calcium pyrophosphate dihydrate deposition in axial skeleton fibrocartilage may be a common finding in patients undergoing repeat spinal surgery.

Cartilage matrix in hereditary pyrophosphate arthropathy.

Morphological and biochemical studies of articular cartilage were performed in 6 members of 3 families with hereditary pyrophosphate arthropathy. Evidence of metabolic disturbance of cartilage matrix was obtained from light and electron microscopic findings and by the content and composition of glycosaminoglycans.

Calcium pyrophosphate dihydrate deposition disease and familial hypomagnesemia.

A 40-year-old woman presented with calcium pyrophosphate synovitis and chondrocalcinosis. She was subsequently found to have hypomagnesemia, as did her 22-year-old son. Metabolic studies demonstrated normal gastrointestinal absorption of magnesium, and impaired renal conservation of magnesium without other evidence of renal tubular dysfunction. It seems likely that a genetically determined abnormality of magnesium metabolism was responsible for the occurrence of chondrocalcinosis in this patient.

Calcium pyrophosphate crystal formation in model hydrogels. II. Hyaline articular cartilage as a gel.

We studied calcium pyrophosphate crystal formation in an in vitro cartilage system. Two parallel troughs were excavated in tibial plateau articular cartilage obtained postmortem. One well was filled with solid sodium pyrophosphate, the other with calcium chloride. After incubation for 24 h at either 10 degrees C or 37 degrees C the precipitate band between the troughs was analyzed for the size and nature of crystals present. In subsequent experiments, the cartilage was pretreated by laceration, contusion, trypsin or hyaluronidase denaturation. We found that cartilage denaturation resulted in formation of larger crystals but that the crystal product in all experiments was identical, alpha CaNa2P2O7.4H2O a nonphysiologic crystal.

Pyrophosphate and diphosphonates in skeletal metabolism. Physiological, clinical and therapeutic aspects.

Pyrophosphate and diphosphonates produce striking results on calcium metabolism in experimental animals and man. Compounds containing P-O-P- bonds (e.g. inorganic pyrophosphate [PP-ii1 or P-C-P bonds (diphosponates) inhibit both the formation and dissolution of calcium phosphate crystals in vitro. PP-i may have a physiological function in regulating calcification and bone turnover, and obnormalities in its metabolism may occur in some human diseases notably hypophosphatasia and pseudogout. Diphosphonates inhibit ectopic calcification, and slow down resorption and bone turnover in several experimental systems in vivo. They have helped in studies of various aspects of the regulation of calcium metabolism. The diphosphonate, disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) has been shown in clinical studies to be effective against ectopic calcification particularly in myositis ossificans progressiva and in disorders of increased bone resorption such as Paget's diseases and some types of osteoporosis. -99mTechnetium complexes of EHDP, PP-i and other polyphosphates have also recently been used successfully as bone scanning agents.