Ann Rheum Dis: first published as 10.1136/ard.42.Suppl_1.27 on 1 January 1983. Downloaded from

Ann Rheum Dis (1983), 42, Supplement p 27

Pathogenesis of chondrocalcinosis and pseudogout. Metabolism of inorganic pyrophosphate and production of calcium pyrophosphate dihydrate crystals

A. CASWELL, D. F. GUILLAND-CUMMING, P. R. HEARN, M. K. B. McGUIRE, R. G. G. RUSSELL From Department of Human Metabolism and Clinical Biochemistry, University ofSheffield Medical School, Beech Hill Road, Sheffield S10 2RX.

Introduction General considerations pseudogout." '" Here there is an enzyme defect, a deficiency of alkaline Calcium pyrophosphate dihydrate Table 1 summarises the factors to be phosphatase with resultant rise of (CPPD) is one of several types of considered in relation to the inorganic pyrophosphate (PP,) in body crystal that may be deposited in the deposition of CPPD crystals and the fluids. Hypophosphatasia is the best body. The term chondrocalcinosis is association of chondrocalcinosis with example of how an abnormality in

used to describe the radiographic endocrine and metabolic diseases. The pyrophosphate metabolism may by copyright. appearance of calcified deposits, reasons for these clinical associations contribute to the production of notably in the articular cartilage and are not always clear but they indicate chondrocalcinosis, but raises the menisci of the knees but also in other that a variety of metabolic disturbances intriguing question of why CPPD .' Not all calcified deposits within may lead to deposition of crystals appear in the typical sites in joints are due to calcium pyrophosphate crystals. cartilage, even though PP, pyrophosphate; however, occasional CPPD deposition is also more concentrations are raised deposition of apatite,2 calcium common in the presence of other systemically. dihydrogen phosphate,' and oxalate disease, including osteoarthropathy,5 It is apparent that chondrocalcinosis also occurs. neuropathic (Charcot) joint disease,7 must be considered a multifactoral McCarty was the first to identify and .5 In such cases the destructive problem in which several metabolic CPPD crystals in synovial fluids taken changes that occur may induce and physiochemical factors probably from patients thought to have gouty alterations of pyrophosphate interact to produce CPPD crystals. http://ard.bmj.com/ ,"5 He found the crystals to be metabolism within the joint leading to Before considering the mechanisms of the calcium pyrophosphate dihydrate increased crystal formation. crystallisation involved in production salt in its triclinic form. Shedding The increased incidence of CPPD of CPPD crystals, we will first review crystals into the synovial space deposition with aging may also be current knowledge of pyrophosphate produces acute or chronic attacks of related to the changes that occur in metabolism. pseudogout.6 cartilage with age. Certain considerations apply to all A familial form of chondrocalcinosis

Intracellular metabolism of inorganic on September 27, 2021 by guest. Protected forms of crystal deposition, in was described by Zitnan and Sit'aj in pyrophosphate particular the factors determining 1958," although at that time it was whether or not crystallisation occurs. not known that CPPD was the calcium This largely concerns the activity salt responsible. Subsequently other GENERAL CONDITIONS products of the ions involved, although familial forms have been Inorganic pyrophosphate (PP,) is the presence of nucleating agents or described.'0"'- The existence of these produced at one or more steps in a inhibitory agents, or both, must be inherited forms allow a comparison wide variety of biochemical pathways considered. Solubilisation processes with classic gout where specific that lead to the synthesis of most of the may also be important. enzyme defects have occasionally been major cell constituents. Hence The purpose of this presentation is identified--for example, in the generation of PPi occurs during the twofold: (a) to review current Lesch-Nyhan syndrome.'3 The biosynthesis of proteins, lipids, knowledge of the metabolism of underlying metabolic defect in the phospholipids, nucleotides, and inorganic pyrophrosphate and (b) to familial forms of chondrocalcinosis is nucleic acids, urea, steroids, structural describe some of the mechanisms that still not determined but may entail polysaccharides, and glycogen. may be involved in the production of enzyme abnormalities. Breakdown of pyrophosphate is CPDD crystals, both in vitro and in Hypophosphatasia is an inherited brought about by a hydrolysis reaction vivo. disorder associated with catalysed by inorganic Ann Rheum Dis: first published as 10.1136/ard.42.Suppl_1.27 on 1 January 1983. Downloaded from

Suppl p 28 Annals ofthe Rheumatic Diseases

Table 1 Conditions associated with deposition ofcrystals ofcakium pyrophosphate dihy- concentration of intracellular PPi drate (CPPD) in joints (chondrocalcinosis). Note the large number ofpossible underlying would be under metabolic control and mechanisms. could respond to changes in the rates of either its synthesis or degradation. Possible mechanisms involved Furthermore, if the intracellular Inherited forms: concentration of PP1 does change in Described from Possible abnormality in inorganic pyrophosphate response to different metabolic Czechoslovakia, Chile, Netherlands, (PP,) metabolism-for example, over- conditions, it becomes possible for this Sweden, France, USA production of PP, or decreased degradation of ion itself to be involved in the PP1 (possible changes in pyrophosphatases). regulation of metabolism. Reduced inhibitors of crystallisation. General associations: Aging Disturbed PP1 metabolism. IS THE PPi HYDROLYSIS Physical damage to chondrocytes. REACTION IN EQUILIBRIUM? Release of nucleating agents or decreased Attempts to determine the inhibitory activity (perhaps via action of released equilibrium constant and free energy proteases to destroy inhibitors). Increased change for hydrolysis of PP, under cartilage permeation by Ca"+ and PP1. stimulated physiological conditions Other joint disease: have produced variable results.20 22 Epitaxy on apatite crystals. Neuropathic joint disease pH may be lowered during inflammation which However, the reported tissue (Charcot joints) promotes crystal transformations to more concentrations of PPi appear to exceed Destructive insoluble forms. those calculated from values found for Ochronosis the equilibrium constant,'8 19 23 supporting the view that the hydrolysis Urate gout Epitaxy on urate crystals reaction is not in equilibrium. Using Metabolic disorders: freeze-clamped rat tissues we have Hyperparathyroidism Raised extracellular Ca, and/or raised PP1 due to increased adenylate cyclase activity. recently demonstrated that the total by copyright. Hypothyroidism Metabolic changes in cartilage PP, content of skeletal muscle (50 Hypophosphatasia Raised PP1 due to alkaline phosphatase deficiency nmol/g) is substantially higher than Haemochromatosis Fe as nucleating agent or that found in liver, kidney, heart, and pyrophosphatase inhibitor lung tissues (20 nmol/g) (unpublished Long-term steroid therapy Secondary hyperparathyroidism observations). Similarly, with isolated Possible associations: human cells, it has been observed that Hypertension fibroblasts and synovial cells contain Renal insufficiency High PP, in chronic renal failure; acidosis promotes than and bone crystal transformation to insoluble forms less PP1 chondrocytes Metabolic alterations in cartilage cells. Furthermore, the higher PP1 Paget's disease Age related content of chondrocytes relative to

Diabetes mellitus fibroblasts appears to be correlated http://ard.bmj.com/ Wilson's disease Copper as crystal nucleating agent or with differences in the rate of pyrophosphatase inhibitor proteoglycan synthesis in these two cell types.24.26 Several studies have shown that both the total PP1 content and the calculated cytoplasmic free PP1 pyrophosphatases, in which 2 mol of hydrolysis reaction for PP1 is at content of freeze-clamped rat liver orthophosphate (Pi) are produced per equilibrium in vivo. An alternative may change quite considerably after mol of PP1 cleaved. Such enzymes possibility may be that inorganic various short term or long term on September 27, 2021 by guest. Protected include glucose- 6-phosphatase and pyrophosphatase is a nonequilibrium metabolic manipulations-for alkaline phosphatase, as well as more enzyme and that its activity is the example, after administration of specific inorganic pyrophosphatases. limiting factor in the removal of PP1. acetate or butyrate or after 48 hours' The metabolic importance of PP1 Additional determinants of the starvation."9 21 23 Such studies have has yet to be defined. The assumption intracellular steady state led to the suggestion that hepatic that intracellular concentrations of PP1 concentration of PP1 may be the glucose uptake and phosphorylation are very low led Kornberg and others'6 intracellular concentration of Pi, both are regulated predominantly by 1 to point out that the removal of PP, as a result of the effect of Pi on the changes in the concentration of free provides a means of driving equilibrium reaction, and because it is PPi in cytoplasm,2' thereby indicating pyrophosphorylase reactions in the a competitive inhibitor of a role for PP1 in metabolic regulation. direction of synthesis, essentially pyrophosphatases. If the An increase in the intracellular rendering them irreversible. However, pyrophosphatase reaction is not in concentration of PP1 in response to the observations of detectable amounts of equilibrium, the concentration of PPi stimulation of a biosynthetic pathway PPi in rat liver, for example,'8 '9 will be determined by the balance producing PP1 has also been observed question this assumption, and, between the rate of formation and in isolated human articular moreover, question whether the breakdown of PPi. In this case, the chondrocytes after enhancement of Ann Rheum Dis: first published as 10.1136/ard.42.Suppl_1.27 on 1 January 1983. Downloaded from

Pathogenesis ofchondrocalcinosis and pseudogout Suppl p 29 glycosaminoglycan synthesis by Inorganic pyrophosphatase appears little information about the movement treatment with xylosides.2" to be principally located in the of PPi across cell membranes or about Raised intracellular concentrations cytosol.a 32 Smaller amounts of the which organs make appreciable of PPi have also been reported in activity occur in mitochondria.33 contributions to the PP, content of isolated fibroblasts and lymphoblasts Inorganic pyrophosphatase activity extracellular fluid. There is evidence derived from patients of a French has also been demonstrated in that this PP, is of endogenous origin; family with an hereditary form of endoplasmic reticulum in some tissues, thus it is not derived direct from the chondrocalcinosis.12 27 This but this may be due to the presence of a diet, as dietary PP1 and observation raises the question of glucose-6-phosphatase,4 35 which also polyphosphates appear to be whether some of the hereditary forms possesses pyrophosphatase activity. completely hydrolysed to Pi within the of chondrocalcinosis are associated Studies of pyrophosphatase in the intestinal lumen, probably by the with overproduction of PP, in cells, cytosol from various tissues (red blood action of alkaline phosphatase, which due to an abnormality in one of the cells, polymorphonuclear leukocytes, is present in the brush border biosynthetic pathways generating PP,. cartilage, dental pulp, etc.) have membranes of enterocytes.42 Reports of abnormal cartilage matrix suggested that this enzyme is specific Studies of PP, turnover have been production in patients from two for PPi31 32 ...39 has a pH optimum of restricted to the examination of Swedish families with a hereditary 7-8, requires Mg++ for activity, is extracellular PP,. Studies using form of this disease might support this strongly inhibited by other divalent 32P-labelled PPi indicate that plasma view.'2 28 metal cations even in the presence of PP, turns over extremely rapidly in Hence evidence appears to indicate Mg++-for example, Ca++, Fe", and dogs and in man.4" In dogs the that the intracellular concentration of Cu'' and is also inhibited by fluoride hydrolysis to Pi accounts for at least PP, does vary with such factors as cell ions. Several known inhibitors of 25% of the loss of PP1 from the plasma type, metabolic conditions, and the alkaline phosphatase-for example, compartment whereas urinary presence of disease states, supporting Pi, imidazole, and CN- have little excretion accounts for only 10%.43 the hypothesis that the hydrolysis inhibitory effect on this enzyme and Hence hydrolysis to Pi would appear to reaction is not in equilibrium and that this, together with its specificity, be a major mechanism for the removal the concentration of this ion is under suggest that this inorganic of PP, from the plasma compartment. by copyright. metabolic control. pyrophosphatase activity is a function 32P-labelled PP1 added to whole blood of a discrete enzyme and is not simply in vitro, however, is only relatively CONTROL OF THE a function of alkaline phosphatase. slowly hydrolysed, implying that the INTRACELLULAR Reports of the Km for PP1 of a major hydrolytic enzymes are not CONCENTRATION OF PPi cytosolic inorganic pyrophosphatase circulating but are located on or within Factors involved in the control of the are in the range 11-40 mmol, which cells.43 intracellular PP, concentration have may imply that the enzyme is not yet to be defined. As discussed above, saturated with substrate under ORIGIN OF EXTRACELLULAR PPi PPi production will be controlled by physiological conditions. IN ARTICULAR CARTILAGE those factors which regulate Mitochondrial inorganic If CPPD crystals are first formed biosynthetic pathways containing PPi pyrophosphatase activity occurs in two outside cells rather than inside, then http://ard.bmj.com/ generating steps and it will be forms, one of which is membrane the origin of extracellular PP, and necessary to delineate the relative bound. Both forms resemble the mechanisms controlling the local contribution of the different pathways cytosolic activity in that they are concentrations of this ion become to the overall rate of PP, production in specific for PPi, require Mg++ for important. (Fig. 1) the cell. PP, breakdown will be activity, have pH optimum of 7-8 and The PP, present in the extracellular controlled by those factors which are inhibited by divalent cations-for space of articular cartilage could either regulate inorganic pyrophosphate example, Ca++-and by fluoride.334 4 arise from the intracellular compart- The effects of and be activity. Mg++ Ca++ may ment or it could be synthesised on September 27, 2021 by guest. Protected of particular relevance and we are at extracellularly or released from sub- the influence of the TISSUE DISTRIBUTION AND present examining chondral bone. Release of PP, from of these cations on the PROPERTIES OF INORGANIC availability the intracellular compartment could PYROPHOSPHATASE intracellular concentration of PP, in occur by a variety of mechanisms--for A recent study of inorganic human articular chondrocytes and example, by a specific membrane car- pyrophosphatase activity in the rat bone cells. rier for PP,, in conjunction with the showed that the total activity varies secretion of matrix components or fol- quite considerably between tissues and Extracellular metabolism of inorganic lowing cell damage or death. that the total hepatic activity changes pyrophosphate during development.2' These results THE RELEASE OF PP1 FROM imply that the total tissue activity of GENERAL CONSIDERATIONS ARTICULAR CARTILAGE this enzyme is regulated, but it should Although PPi occurs extracellularly in Generation of PPi in vitro by cartilage be noted that the activities measured body fluids-for example, serum, fragments was first reported by in this study under optimal conditions plasma, urine, saliva, and synovial Howell's group,45 who stated that PPi in vitro do not necessarily reflect the fluid, and large quantities also occur was released from growth plate real activities in vivo. adsorbed to bone mineral, there is cartilage and from articular cartilage Ann Rheum Dis: first published as 10.1136/ard.42.Suppl_1.27 on 1 January 1983. 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Suppl p 30 Annals ofthe Rheumatic Diseases

phosphate-buffered saline or medium without serum, no detectable release INTRACELLULAR EXTRACELLULAR of PP, from these cells occurs. SPACE SPACE Furthermore, when the concentration of PPi in the medium is raised to around 100 ,.tmol/l rapid hydrolysis of PP; occurs.24 47 However, using a more sensitive assay, we have recently been able to demonstrate the release of small amounts of PP, from washed cAMP monolayers of human articular 2P. chondrocytes incubated in medium without serum. Additionally, we have observed that under our conditions, in which the extracellular PP, Pos release of concentration is generally less than 1 extr proteoglycans plus PP i ,tmol/l PP1 hydrolysis determined with in vesicles 32P-labelled PPi in the medium is very slow (A. M. Caswell and others, p. 99). This difference between our results Nucleoside triphosphate leakage ? and those of others47 may imply the (NTfP) - presence of an extracellular inorganic cell damage ? BLoaynthetic reactions pyrophosphatase activity with a eg proteoglycan synthesis relatively high Km for PP,. 7 These various results suggest that NUCLEOSIDE TRIPHOSPHATE NUCLEOSIDE TRIPHOSPHATE P YROPHOSPHORYIASES PYROPHOSPHOHYDROLASE chondrocytes possess the ability to (ectoenzyme) release limited amounts of PP,. In this by copyright. context, it may be relevant that it has not been possible to show the passage + PPI of PPi across the membrane of the red ~~~/ * PPi cell.46 It is also of interest that, in the study of McCarty's group described PYROPHOSPRATASE release of from rabbit (ectoenzyme) above, PPi eg alkaline cartilage fragments, but not from phosphatase human cartilage fragments, was positively correlated with release of 2PI uronic acid.46 However, this does not necessarily establish that PP, and http://ard.bmj.com/ uronic acid are released from the cell 2Pi together since, as suggested earlier, changes in the rates of synthesis of matrix components could result in Fig. 1 Pyrophosphate metabolism: a schematic representation ofpossible parallel changes in intracellular PP,. sources ofboth intracellular and extracellular PPi. PPi introduced in the Such changes could in turn influence intracellular compartment may be either co-secreted with products ofintracellular the rate of release of PPi from the cell biosynthetic reactions-for example, proteoglycans-or may leak to the by any putative carrier mechanisms on September 27, 2021 by guest. Protected extracellular compartment when cells are damaged, as happens in degenerative specific for PPi. Release of PP, into the joint disease. Extracellular PP, may arise from the activity ofthe ectoenzyme, extracellular space of cartilage after nucleoside triphosphate pyrophosphohydrolase, acting presumably on nucleoside damage or death of chondrocytes may occur and could explain why.patients triphosphates 'leaked' to the extracellular compartment. who have had meniscectomies have a higher incidence of chondrocalcinosis in the operated knee than in the derived from young rabbits and from of PPi from fragments of articular unoperated knee.49 The extent to patients with osteoarthritis but not cartilage and fibrocartilage derived which this release mechanism occurs in from rabbit ear cartilage or from from both young and adult rabbits and from of 'normal' human undamaged cartilage or in other articular cartilage derived from fragments 'normal' tissues is not known. mature rabbits or 'normal' humans. articular cartilage. Extrusion of PP, However, in a recent study, McCarty's may therefore be a feature common to group,4" using a highly sensitive assay all types and.age of cartilage. PRODUCTION OF PPi and correcting for PP1 hydrolysis It has been reported that, if washed EXTRACELLULARLY during the incubation by.the use of monolayers of human articular Another possible origin of 32P-labelled PPi, have observed release chondrocytes are incubated in extracellular PP, is that it is generated Ann Rheum Dis: first published as 10.1136/ard.42.Suppl_1.27 on 1 January 1983. Downloaded from

Pathogenesis ofchondrocalcinosis and pseudogout Suppl p 31 outside the cell by membrane bound guanosine triphosphate, cytidine would be necessary so that PP, could enzymes. One such enzyme is adenylate triphosphate, and uridine reach subchondral bone through the cyclase which catalyses the reaction: triphosphate. These preliminary blood supply on the metaphyseal side observations suggest that presence of a and then subsequently be released ATP---3' ,5'-cyclic AMP + PP,. highly active nucleoside triphosphate down a concentration gradient This enzyme is located in cell pyrophosphohydrolase on the outside towards the articular surface. This membranes and is activated in of the plasma membrane of human could account for the observation that response to hormone and other articular chondrocytes with an calcium pyrophosphate dihydrate agents-for example, certain drugs.50 apparently low Km for nucleoside deposits in the mid zone of articular The cyclic AMP formed is released triphosphates. The function of this cartilage, since this would be the point into the intracellular compartment but activity in chondrocytes is unclear but at which PP1 from the subchondral the fate of PP, formed is unknown and could be similar to that suggested bone and calcium from the joint space it is not known whether any is released earlier for nucleoside triphosphate would come together.56 into the extracellular fluid. pyrophosphohydrolase activities in Another enzyme of considerable other tissues-for example, a role in ALKALINE PHOSPHATASE AND interest is nucleoside triphosphate calcium transport. REMOVAL OF PPi FROM THE pyrophosphohydrolase, which It is therefore interesting that the EXTRACELLULAR SPACE catalyses the reaction: total concentration of nucleotides in As in the case of intracellular PP, NTP--NMP+ PP,, where NTP de- the extracellular fluid of chicken metabolism, the major mechanism for notes nucleoside triphosphate and epiphyseal cartilage has been reported removing PPi from the extracellular NMP nucleoside monophosphate. to range from 200 ,u mol/l in the compartment is enzymatic hydrolysis. In rat liver the enzyme is located in proliferating zone to 740 molIl in the The addition of inorganic the plasma membrane, is calcium hypertrophic zone.55 Thus there is a pyrophosphatase, to extracellular dependent and hydrolyses both potential mechanism for the fluids--for example, plasma-results in pyrimidine and purine nucleoside extracellular generation of PPi, at least a pronounced reduction in the PP1 triphosphates with Kms in the Molar in growth cartilage, since appreciable concentration and therefore the range.5"-53 The enzyme has been amounts of PP, could be generated if reaction: by copyright. proposed to function either in calcium nucleoside triphosphates comprised PP 2Pi transport52 or together with even a small fraction of the observed cannot be in equilibrium and the 5'-nucleotidase in the salvage of total nucleotide concentration. It is also interesting to note that in breakdown of extracellular PP, must nucleoside triphosphates that leak Howell's be limited by the activity of hydrolytic from the cell.53 In rats this enzyme has the recent study by group enzymes. been shown to exhibit a wide tissue described above,54 nucleoside The physiological importance of distribution and the activity appears to triphosphate pyrophosphohydrolase in the activity was greater in articular alkaline phosphatase activity vary quite considerably with tissue derived extracellular hydrolysis of PP; has type, the highest activities being cartilage homogenates from been demonstrated mainly by studies observed in liver, small intestine, and patients with chondrocalcinosis than in of patients with hypophosphatasia in

from http://ard.bmj.com/ kidney and much lower activities being those derived patients with whom a deficiency in alkaline observed in brain, thymus, and osteoarthritis. One sample derived phosphatase activity occurs."4 There blood.52 from a 'normal' patient contained no is a fourfold increase in the plasma PP1 The presence of a virtually activity. This apparent with a nucleoside increase in the in a situation in concentration corresponding triphosphate pyrophosphohydrolase activity reduction in the rate constant of activity in human articular cartilage which increased amounts of PP1 occur hydrolysis in the removal of PP, from has been inferred from a recent extracellularly supports the view that study this enzyme is important in the the extracellular compartment.5 4 5 of Howell's group, who demonstrated generation of PP, extracellularly in Such observations establish that the generation of PP, after adding 1 alkaline phosphatase does function as on September 27, 2021 by guest. Protected mmol/l ATP to cartilage articular cartilage. Furthermore, there may be a role for this activity in the a pyrophosphatase in vivo and that is homogenates.54 However, the use of pathogenesis of chondrocalcinosis an ectoenzyme capable of hydrolysing homogenates precludes any statement after substrates present in the extracellular about the location of this enzyme cartilage damage, since the compartment. It can be calculated that or the leakage of metabolites from disrupted activity number of possible cells could result in the release -alkaline phosphatase may be activities involved. of responsible for the removal of as much We have been able to demonstrate nucleoside triphosphates, which could then serve as substrates for the as 80% of the PPi delivered to the the generation of substantial amounts extracellular compartment in normal of PPi extracellularly after adding ATP generation of PP,. individuals. (concentration range 6-25-400 Amol/1) to washed human articular RELEASE OF PPi FROM PROPERTIES OF THE PPj chondrocyte monolayers incubated in SUBCHONDRAL BONE HYDROLYTIC ACTIVITY OF medium without serum (A. M. Caswell Local release of PP, from subchondral ALKALINE PHOSPHATA-SE and others, p. 00). The effect can also bone has been suggested as a further The properties of the hydrolytic be observed with other nucleoside possible source of extracellular PP, in activity of alkaline phosphatase triphosphates-for example, articular cartilage. Some polarity towards PP1 has been studied in a Ann Rheum Dis: first published as 10.1136/ard.42.Suppl_1.27 on 1 January 1983. Downloaded from

Suppl p 32 Annals ofthe Rheumatic Diseases variety of tissues-for example, liver, Pi, and further studies are needed to haemodialysis or renal transplant.76 ' kidney, and small intestine,56 determine whether such agents An increase in the PP, content of bone bone6' 62 and epiphyseal cartilage and regulate the breakdown of has also been noted in some patients matrix vesicles.63-' This enzyme exhibits extracellular PP, in vivo. with chronic renal failure and it has remarkably similar properties in a been suggested that a relationship wide variety of tissues. The pH IS A DEFECT IN CLEARANCE OF exists between the bone content of PP, optimum of the PP, hydrolytic activity PPi FROM ARTICULAR and the extent of soft tissue decreases with decreasing PP1 CARTILAGE INVOLVED calcification in these patients.79 concentration, so that at physiological IN THE PATHOGENESIS OF The plasma concentration of PP, has concentrations of PPi approaches pH CHON DROCALCINOSIS? been reported to be raised in some 7- 0. Magnesium ions affect the activity There is little direct evidence for a cases of acromegaly and this increase in a complex manner and this effect defect in PP, clearance from articular appears to be correlated with an appears to be influenced by the cartilage in patients with idiopathic increase in the plasma Pi availability of substrate with an chondrocalcinosis. There have been concentration.' "' Plasma PP, is also optimal activity occurring at a reports that inorganic pyrophosphatase raised in some patients with Mg+/PPi concentration ratio of 1: 1. activity due to both alkaline osteomalacia due to vitamin D This led to the suggestion thalt the true phosphatase and glucose-6- deficiency, but apparently not in some substrate for the enzyme is MgP2O72- phosphatase is reduced in joint of the inherited forms of vitamin and that this cannot form if PP1 is fluids from patients with D-resistant renal tubular rickets, nor present in excess over Mg++, whereas if chondrocalcinosis.66... In other in osteomalacia associated with total Mg++ is present in excess, MgP2O7 studies, however, no change was parenteral nutrition,'5 (M. K. B. forms, which is inhibitory. In the observed either in the hydrolytic McGuire and others, paper presented presence of Mg++, Ca++ is only slightly activity of alkaline phosphatase at 14th Annual Meeting of American inhibitory but the activity is subject to towards PP, or in an inorganic Society of Nephrology, 1981). inhibition by substrate and product pyrophosphatase activity, with an acid A defect in urinary PP, excretion (Pi). The Km of alkaline phosphatase pH optimum, in joint fluids from these may contribute to formation of renal for PPi inhibition has been reported to patients.70 Moreover, as noted earlier, stones in some cases. In several studies by copyright. range from 40-85 molIl but some of Howell's group observed an increase it has been reported that urinary PP1 these differences may be accounted for rather than decrease in the PPi excretion is reduced in men who form by the use of different concentration hydrolytic activity of alkaline stones but not women, with this effect ratios of Mg'+ to PP1. phosphatase in articular cartilage being most marked in the 30-40 age In a recent study, Howell's group54 derived from patients with group."-83 have extracted and examined the chondrocalcinosis.54 A defect in extracellular PPi properties of alkaline phosphatase However, some of the disease metabolism is unlikely in osteogenesis derived from adult human articular associations observed suggest that imperfecta as we have been unable to cartilage. Virtually no activity could be there is a defect in PP, clearance confirm the observation of Solomon's obtained from 'normal' samples but from articular cartilage at least in some group that the serum PPi appreciable amounts of the activity cases of chondrocalcinosis-for concentration is raised in this http://ard.bmj.com/ were obtained from patients with example, in hypophosphatasia.7' disorder."5 chondrocalcinosis and even higher Similarly, the associations between In this context it is important to note activities were obtained from patients chondrocalcinosis and hyperpara- that measurements of PP, in serum with osteoarthritis. thyroidism,7274 haemochromatosis7' give values two to four-fold higher In articular cartilage derived from and hypomagnesaemia73 could than in plasma. This is due to the patients with osteoarthritis or reflect the influence of Ca++, Fe++, release during blood clotting of PP, chondrocalcinosis, there appear to be and Mg++ respectively on the pyro- stored in the dense granules of two forms of alkaline phosphatase, phosphatase activity of alkaline phos- platelets. on September 27, 2021 by guest. Protected both of which possess hydrolytic phatase. There is little evidence for any -activity towards PP, but whereas one change in the plasma PP, form of the enzyme resembles the concentration in either rheumatoid CLINICAL CONDITIONS IN WHICH arthritis or activity found in other tissues, the DISORDERS osteoarthritis.'7 8 86-86 OF EXTRACELLULAR In summary, there to be no other appears to be activated rather PP1 METABOLISM OCCUR appears than inhibited when Mg` is in excess Measurements of PP, in serum, systemic disorder in PP1 metabolism over PPi. It is not known, however, plasma, or urine in a variety of clinical associated with most cases of whether both forms of the enzyme are conditions have suggested that chondrocalcinosis. found in normal adult human articular disorders of extracellular PP, cartilage. metabolism do occur in some disease Physicochemical studies of calcium Taken together, these studies of the states, hypophosphatasia being the pyrophosphate crystal formation hydrolytic activity of alkaline best example. However, plasma and phosphatase towards PP, demonstrate serum PPi concentrations are also Little is known about the that this activity can vary in response raised in about one third of patients physicochemical conditions necessary to a variety of physiological with chronic renal failure, and the for the formation of calcium agents-for example, Mg++, Ca,- and values return to normal after pyrophosphate dihydrate (CPPD) Ann Rheum Dis: first published as 10.1136/ard.42.Suppl_1.27 on 1 January 1983. 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Pathogenesis ofchondrocalcinosis and pseudogout Suppl p 33 crystals in articular cartilage and other concentration required for crystal experiments. At 40 4g/ml the added sites in vivo. The problem can be initiation was lowered to 75 molIl. This crystals raised the amount of considered from several points of reflects conditions which are the pyrophosphate required to initiate view: closest to physiological that have so far CPPD crystal growth, presumably (a) What factors are necessary for been explored. It appears, therefore, explained by their ability to absorb initiating crystal deposition? (b) What that the formation of CPPD crystals in pyrophosphate, thereby making it determines the interconversions of synovial fluid would not be favoured in unavailable. At lower concentrations different crystal forms of CPPD? (c) vivo. To explain where and why (4,g/ml) preliminary results suggest What influences the formation of the crystals form in the body one therefore that hydroxyapatite crystals are crystals, their solubility and removal needs to invoke additional without effect, suggesting a balance from the joint? mechanisms such as increased local between nucleating effects and surface concentrations of either calcium or adsorption of pyrophosphate. Further INITIATION OF CRYSTAL PP,, or the presence of nucleating work is required to clarify this point. DEPOSITION agents. The formation of CPPD crystals in The concept of nucleating CRYSTAL TRANSFORMATIONS pathological conditions may be mechanisms leading to crystal The type of CPPD crystals found promoted in various ways. Firstly, the formation is an attractive one which naturally in vivo are predominantly in concentrations of calcium or PPi may requires further study. The association the triclinic form, although McCarty et be raised; the increased of pyrophosphate arthropathy with al94 and Bywaters et al95 have reported concentrations of PP, could result from haemochromatosis7" was investigated the occurrence ofthe monoclinic form. either enhanced production or and the possibility that iron salts might Crystals produced in the three day decreased removal from the joint, as, act as nucleating agents for crystal incubations under the experimental for example, in hypophosphatasia." "712 formation was tested in the crystal conditions of Hearn and Russell' 90 By analogy, increased plasma calcium growth system in vitro. The presence were shown by x-ray diffraction to be concentrations occur in of low concentrations of ferric salts orthorhombic in the absence of Mg-+ hyperparathyroidism, although (Fe++ at 25 umol/l) promoted crystal and amorphous in the presence of whether or not the association with growth with the result that the amount physiological concentration of Mg+. by copyright. deposition of CPPD crystals is of PPi needed for crystal formation Longer incubations of one month or mediated via raised calcium was reduced to one quarter of that more with added magnesium appear to concentrations is at present unknown. required in the absence ofiron.89 It was allow the slow formation of crystals of Crystal formation might also be thought that this effect may have been the monoclinic type, followed by a favoured by the presence of nucleating due to the colloidal nature of Fe-+ salts slow transition to the triclinic form. agents or by the removal of any neutral in solution at neutral pH. Urate at The presence of 1 mmolI phosphate inhibitors of crystal formation, physiological concentrations also has a considerably increased the rate of this although currently very little is known small promoting effect on the transition, but even then failed to about these possible mechanisms. In precipitation of calcium convert a significant proportion of order to study some of these questions pyrophosphate'9 but it is doubtful crystals to the triclinic form. whether this effect is we have devised a simple method to potent enough to The use of nucleating agents has so http://ard.bmj.com/ define the conditions necessary for explain the association found between far failed to influence the type of crystal deposition in vitro.89 90 urate gout and pyrophosphate crystal formed from solution. The formation of crystals was arthropathy. Hydroxyapatite crystals promote studied in simple synthetic solutions Crystal formation in vitro increases CPPD crystal formation but the types which mimic extracellular and synovial rapidly as the pH rises through the formed are orthorhombic and fluids. At physiological concentrations range of 7 2 to 7-4 suggesting that in monoclinic. Similarly, addition of of calcium (1- 5 mmol/) at pH 7- 4 and vivo a small change in pH could induce preformed CPPi crystals of these types ionic crystal formation without such large physiological strength, crystals of leads only to further growth of those on September 27, 2021 by guest. Protected calcium pyrophosphate were found to changes in the concentration of Ca and crystals and new growth of similar form within three days at 370C, when PP, being required. These results are crystals, with no increase in transition the PPi concentration was 40 ,umol/l or interesting in relation to studies of to the triclinic forms. higher. In the presence of Mg++ at Howell and Pita's group,9'" who have These studies are based on physiological concentrations (0.5 recorded high pH in extracellular fluid microscopical identification and await mmolIl), crystals formed only when aspirates from epiphyseal cartilage by confirmation by x-ray diffraction the PP, concentration reached 175 micropuncture techniques. The pH of techniques. Work from the soil mol/l. This contrasts with the fluid within articular rather than chemists of the Tennessee River concentrations of PP, found in normal epiphyseal cartilage is, however, not Valley Authority,96 who investigated synovial fluids (mean 3 molIl; range 1 known. In our studies of crystal calcium pyrophosphates in relation to to 4 mol) and in pseudogout fluid formation in vitro a high pH promoted their use as fertilisers, provides (mean 20 ,umolVl; range 5 to 60 crystal formation. valuable information about the ,umol/l). In the presence of The possible role of nucleation by potential transformations that can physiological concentrations (1 epitaxy has also been explored and occur. A study oftheir results indicates mmol/l) of inorganic phosphate hydroxyapatite crystals were tested for that the monoclinic and triclinic (orthophosphate) the PPi their effect in our crystal growth varieties of CPPD crystals appear to Ann Rheum Dis: first published as 10.1136/ard.42.Suppl_1.27 on 1 January 1983. Downloaded from

Suppl p 34 Annals ofthe Rheumatic Diseases

represent the stable end products of a location of CPPD deposits in articular important in provoking the acute number of potential crystal cartilage and other joint structures inflammatory reaction within the transformations. Regardless of the suggests that there may be some joint. CPPD, as with other crystal physical nature of the first crystals to abnormality of PPi metabolism, types which can provoke deposit, all crystals may therefore metabolite diffusion, or of nucleation inflammation, has been shown to convert ultimately to monoclinic and at these sites. PPi concentrations are induce cell lysis. This may be the basis triclinic varieties in vivo. raised in the synovial fluid but not in for the findings of chondrocyte These studies shed light on the the plasma of patients with lacunae in the vicinity of CPPD mystery ofwhy the naturally occurring chondrocalcinosis and osteoarthritis, crystals in articular cartilage.99 This crystals are usually of the triclinic and with chronically symptomatic may be due to the intracellular occasionally of the monoclinic variety. joints.70"6 100 101 It is uncertain whether dissolution of crystals releasing large In an interesting study, Pritzker et this reflects local abnormalities in PP, amounts of calcium and thereby al97 showed that the monoclinic and metabolism or whether it is a result of poisoning the cell. triclinic crystals could be made to form dissolution of CPPD within the joint The question of why only certain within silica and gelatin gels in vitro. tissues. patients with CPPD crystal deposition Their conditions were close to, but did As crystals are not regularly found disease experience attacks of crystal not match, the natural physiological in synovial fluid in osteoarthropathy, synovitis remains to be determined. state. Thus the concentrations of but only in pseudogout, despite the Presumably certain conditions have to calcium and PPi they started with were reported similarity in synovial fluid be met before crystals will be 'shed' considerably in excess of normal and concentrations of PP, levels, it would from the cartilage to provoke an the pH was below 6 0. appear that either the PP, is derived attack. There appears to be an Our own current work also suggests from different sources in the two association of such attacks with that these crystal conversions actually conditions, or that specific major surgical interevention, occur under simulated physiological mechanisms (such as nucleation of especially where there is a significant conditions, but often take a long time, crystal growth) exist in the joints of postoperative decrease in serum which means that the existence of patients with chondrocalcinosis which calcium concentrations (para-

these specific crystal forms in vivo may lead to CPPD crystal formation. thyroidectomy, major surgery in by copyright. simply be an indication ofthe length of The high negative fixed charge the abdomen or thorax, etc).101 Any time available for crystal conversions density of articular cartilage102 may abrupt fall in serum calcium will lower to take place. favour retention of cations. the [Ca] x [PPi] ion product in the Maroudas103 has suggested that extracellular fluid bathing the CPPD cartilage may show some selectivity for crystals and may result in a loosening CRYSTAL FORMATION, these within the SOLUBILITY AND REMOVAL FROM the retention of calcium, which would of deposits lacunae THE JOINT lead to a rise in total calcium due to crystal dissolution. The sudden In cartilage CPPD crystal deposits concentrations within the cartilage. decrease in size of the crystals may appear initially in rims around cells93 The free ionic calcium concentration enhance their release from sites where and the large deposits are associated would, however, be expected to they were hitherto tightly packed. with empty cartilage lacunae remain similar to that in synovial fluid. Other mechanisms for release include http://ard.bmj.com/ indicating chondrocyte death.9" In the However, in pathological conditions, minor surface fractures induced by synovium, in contrast, CPPD crystals bound calcium might be released trauma or wear. are found only in the phagocytic cells during degradation of proteoglycans of the synovial membrane.99 and thereby initiate the formation of We are grateful to the Arthritis and studies labelled CPPD crystals. Rheumatism Council for their support of Experimental using this work. A. Caswell acknowledges the crystals support the concept that these Finally, it is possible that the raised receipt of a research fellowship from the .c..ystal deposits are sequestered from PPi concentrations arise from Medical Research Council.

t,he synovial fluid rather than being dissolution of CPPD crystals. on September 27, 2021 by guest. Protected formed in the synovium itself. However, Camerlain et al104 found that It seems probable, therefore, that crystals incubated in vitro with joint References are formed in fluid showed very little exchange with !CPPD crystals initially 1 McCarty D J. Clinical assessment of articular cartilage, and appear in the 321PPPi. This contrasts with the rapid arthritis. In: Hollander J L, ed. synovial fluid via a process ofshedding turnover of PP, in vivo and suggests Arthritis and allied conditions. 9th ed. rather than growth in situ of new that either the solubility product for Philadelphia: Lea & Febiger, 1979: CPPD crystals. calcium pyrophosphate had already 131-47. Bone is another tissue with a been attained or that one or more 2 Dieppe P A, Huskisson E C, Crocker possible role in formation of CPPD. components of the turnover system in P, Willoughby D A. Apatite deposition Bone lies close to the joint and the PP, vivo were lacking. The problems of disease. A new arthropathy. Lancet be to are however, and 1976; i: 266-8. concentrations might expected solubility complex, 3 Gaucher A, Faur6 G, Netter P, Pourel be high due to the ability of PP, to synthetic crystals behave differently J, Duheille J. Identification des adsorb to hydroxyapatite surfaces. It is from natural ones-possibly because cristaux observes dans les also possible that nucleating agents are of proteins which coat the crystals and destructrices de la located at the sites where crystal impair dissolution. The phagocytosis chondrocalcinose. Rev Rhum Mal growth starts. The preferential of CPPD crystals is probably Osteoartic 1977; 44: 407-14. Ann Rheum Dis: first published as 10.1136/ard.42.Suppl_1.27 on 1 January 1983. Downloaded from

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4 Hoffman G S, Schumacher H R, Paul 19 Veech R L, Cook G A, King M T. phatase in rabbit dental pulp. J Dent H, et al. Calcium oxalate Relationship of free cytoplasmic Res 1977; 56: 1339-1244. microcrystalline-associated arthritis in pyrophosphate to liver glucose content 32 Raja K B, Smith G P, Peters T J. Sub- end-stage renal disease. Ann Intern and total pyrophosphate to cytop- cellular localization and properties of Med 1982; 97: 36-42. lasmic phosphorylation potential. alkaline inorganic pyrophosphatase in 5 Kohn N A, Hughes R E, McCarty D J, FEBS Lett 1980; 117, suppl: 65-72. human polymorphonuclear leuco- Faires J S. The significance of calcium 20 Flodgaard H, Fleron P. Ther- cytes. Clin Chim Acta 1981; 117: pyrophosphate crystals in the synovial modynamic parameters for the hyd- 33-41. fluid of arthritic patients: the rolysis of inorganic pyrophosphate at 33 Mansurova S E, Belyakova T N, pseudogout syndrome.Ann Intern Med pH 7-4 as a function of [Mg2"], [K+] Kulaev I S. The role of inorganic 1962; 56: 738-45. and ionic strength determined from pyrophosphate in energy metabolism 6 Bennett R M, Lehr J R, McCarty D J. equilibrium studies of the reaction. J of isolated mitochondria. BiochemistrY Crystal shedding and acute Biol Chem 1974; 249: 3465-74. (USSR) 1973; 38: 186-87. pseudogout. Arthritis Rheum 1976; 19: 21 Lawson J W R, Guynn R W, Cornell N, 34 Illingworth B, Cori C F. Glucose-6- 93-7. Veech R L. A possible role for phosphatase and pyrophosphatase 7 Jacobelli S, McCarty D J, Wilcox D C, pyrophosphate in the control of hepa- activities of homogenates of liver from et al. Calcium pyrophosphate dihydrate tic glucose metabolism. In: Hanson patients with glycogen storage disease. crystal deposition in neuropathic P W, Mehlman M A, eds. Biochem Biophys Res Commun 1965; joints. Four cases of polyarticular Gluconeogenesis: its regulation in 19: 10-4. involvement. Ann Intern Med 1973; mammalian cells. New York: Wiley, 35 Hefferan P M, Howell R R. Genetic 79: 340-7. 1976: 481-512. evidence for the common identity of 8 Zitnan D, Sitaj S. Natural course of 22 Lawson J W R, Veech R L. Effects of glucose 6-phosphatase, articular chondrocalcinosis. Arthritis pyrophosphatase-glucose phosphot- Rheum 19: pH and free MG2 on the Keq of the 1976; 363-90. creatine kinase reaction and other ransferase, carbamyl phosphate- 9 Zitnan D, Sitaj S. Mnohopecetna glucose phosphotransferase and inor- familiarlia phosphate hydrolysis and phosphate Kalcifikaciz artikularynch transfer reactions. J Biol Chem 1979; ganic pyrophosphatase. Biochim Bio- chrupiek. Bratisl Lek Listy 1958; 28: 254: 6528-37. phys Acta 1977; 496: 431-5. 217-24. 36 McCarty D J, Pepe P F, Solomon S D, 10 Van der Korst J K, Geerards J. 23 Guynn R W, Veloso D, Lawson J W R, Veech R L. The concentration and Cobb J. Inhibition of human erythro- Articular chondrocalcinosis in a Dutch cyte pyrophosphatase activity by cal- by copyright. Pedigree. Arthritis Rheum 1976; 19: control of cytoplasmic free inorganic pyrophosphate in rat liver in vivo. cium, cupric and ferrous ions. Arthritis 405-9. Rheum 1970; 13: 336. Edvinsson Biochem J 1974; 140: 369-75. 11 Bjelle A, U, Hagstam A. 37 Felix R, Fleisch H. Properties of inor- Pyrophosphate arthropathy in two 24 Lust G, Nuki G, Seegmiller J E. Inor- Swedish families. Arthritis Rheum ganic pyrophosphate and proteoglycan ganic pyrophosphatase of pig scapula 1982; 25: 66-74. metabolism in cultured human articu- cartilage. Biochem J 1975; 147: 111-8. 12 Lust G, Faure G, Netter P, Gaucher A, lar chondrocytes and fibroblasts. Arth- 38 Thuillier L. Purification and kinetic Seegmiller J E. Evidence of a general- ritis Rheum 1976; 19: 479-87. properties of human erythrocyte ised metabolic defect in patients with 25 McGuire M B, Colman C H, Baghat N, Mg2"-dependent inorganic pyrophos- hereditary chondrocalcinosis. Arthritis Russell R G G. Radiometric meas- phatase. Biochim Biophys Acta 1978; Rheum 1981; 24: 1517-21. urement of pyrophosphate in cell cul- 524: 198-206. 13 McKeran R 0, Andrews T M, Howell tures. Biochem Soc Trans 1980; 8: 39 Oyama S, Mitsuma M, Furuyama S.

A, Gibbs D A, Chinn S, Watts R W E. 529-30. Separation of inorganic pyrophos- http://ard.bmj.com/ The diagnosis of the carrier state for 26 McGuire M K B, Bayliss M, Baghat N, phatase from alkaline phosphatase in the Lesch-Nyhan Syndrome. Q J Med Colman C H, Russell R G G. rabbit dental pulp. IntJ Biochem 1981; 1975; 44: 189-205. Pyrophosphate metabolism in human 13: 105-7. 14 O'Duffy J D. Hypophosphatasia articular chondrocytes. Ann Rheum 40 Egremovich N V, Volk E S, Baikov associated with calcium pyrophos- Dis 1981; 40: 207-8. A A, Shakhov Y A. Isolation and phate dihydrate deposits in cartilage. 27 Lust G, Faure G, Netter P, Seegmiller primary characterization of two forms Arthritis Rheum 1970; 13: 381-8. J E. Increased pyrophosphate in fib- of inorganic pyrophosphatase from 15 Russell R G G, Bisaz S, Donath A, roblasts and lymphoblasts from bovine heart mitochondria. Biochemis- patients with hereditary diffuse articu-

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