pathogenesis and pseudogout. metabolism of · supplp28 annalsofthe rheumaticdiseases table 1...

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

Pathogenesis of chondrocalcinosis and pseudogout.Metabolism of inorganicpyrophosphate and production of calciumpyrophosphate dihydrate crystalsA. CASWELL, D. F. GUILLAND-CUMMING, P. R. HEARN, M. K. B. McGUIRE,R. G. G. RUSSELLFrom Department ofHuman Metabolism and Clinical Biochemistry, University of Sheffield Medical School, Beech Hill

Road, Sheffield S10 2RX.

Introduction General considerations

Calcium pyrophosphate dihydrate(CPPD) is one of several types ofcrystal that may be deposited in thebody. The term chondrocalcinosis isused to describe the radiographicappearance of calcified deposits,notably in the articular cartilage andmenisci of the knees but also in otherjoints.' Not all calcified deposits withinjoints are due to calciumpyrophosphate; however, occasionaldeposition of apatite,2 calciumdihydrogen phosphate,' and oxalatealso occurs.McCarty was the first to identify

CPPD crystals in synovial fluids takenfrom patients thought to have gouty

arthritis,"5 He found the crystals to bethe calcium pyrophosphate dihydratesalt in its triclinic form. Sheddingcrystals into the synovial space

produces acute or chronic attacks ofpseudogout.6

Certain considerations apply to allforms of crystal deposition, inparticular the factors determiningwhether or not crystallisation occurs.

This largely concerns the activityproducts of the ions involved, althoughthe presence of nucleating agents or

inhibitory agents, or both, must beconsidered. Solubilisation processesmay also be important.The purpose of this presentation is

twofold: (a) to review currentknowledge of the metabolism ofinorganic pyrophrosphate and (b) todescribe some of the mechanisms thatmay be involved in the production ofCPDD crystals, both in vitro and invivo.

Table 1 summarises the factors to beconsidered in relation to thedeposition of CPPD crystals and theassociation of chondrocalcinosis withendocrine and metabolic diseases. Thereasons for these clinical associationsare not always clear but they indicatethat a variety of metabolic disturbancesmay lead to deposition ofpyrophosphate crystals.CPPD deposition is also more

common in the presence of other jointdisease, including osteoarthropathy,5neuropathic (Charcot) joint disease,7and gout.5 In such cases the destructivechanges that occur may inducealterations of pyrophosphatemetabolism within the joint leading toincreased crystal formation.The increased incidence of CPPD

deposition with aging may also berelated to the changes that occur incartilage with age.

A familial form of chondrocalcinosiswas described by Zitnan and Sit'aj in1958," although at that time it wasnot known that CPPD was the calciumsalt responsible. Subsequently otherfamilial forms have beendescribed.'0"'- The existence of theseinherited forms allow a comparisonwith classic gout where specificenzyme defects have occasionally beenidentified--for example, in theLesch-Nyhan syndrome.'3 Theunderlying metabolic defect in thefamilial forms of chondrocalcinosis isstill not determined but may entailenzyme abnormalities.

Hypophosphatasia is an inheriteddisorder associated with

pseudogout." '" Here there is anenzyme defect, a deficiency of alkalinephosphatase with resultant rise ofinorganic pyrophosphate (PP,) in bodyfluids. Hypophosphatasia is the bestexample of how an abnormality inpyrophosphate metabolism maycontribute to the production ofchondrocalcinosis, but raises theintriguing question of why CPPDcrystals appear in the typical sites incartilage, even though PP,concentrations are raisedsystemically.

It is apparent that chondrocalcinosismust be considered a multifactoralproblem in which several metabolicand physiochemical factors probablyinteract to produce CPPD crystals.Before considering the mechanisms ofcrystallisation involved in productionof CPPD crystals, we will first reviewcurrent knowledge of pyrophosphatemetabolism.

Intracellular metabolism of inorganicpyrophosphate

GENERAL CONDITIONS

Inorganic pyrophosphate (PP,) isproduced at one or more steps in awide variety of biochemical pathwaysthat lead to the synthesis of most of themajor cell constituents. Hencegeneration of PPi occurs during thebiosynthesis of proteins, lipids,phospholipids, nucleotides, andnucleic acids, urea, steroids, structuralpolysaccharides, and glycogen.Breakdown of pyrophosphate isbrought about by a hydrolysis reactioncatalysed by inorganic

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Table 1 Conditions associated with deposition of crystals ofcakium pyrophosphate dihy-drate (CPPD) in joints (chondrocalcinosis). Note the large number ofpossible underlyingmechanisms.

Possible mechanisms involved

Inherited forms:Described from Possible abnormality in inorganic pyrophosphateCzechoslovakia, Chile, Netherlands, (PP,) metabolism-for example, over-Sweden, France, USA production of PP, or decreased degradation of

PP1 (possible changes in pyrophosphatases).Reduced inhibitors of crystallisation.

General associations:Aging

Other joint disease:OsteoarthritisNeuropathic joint disease

(Charcot joints)Destructive arthropathyOchronosisRheumatoid arthritisUrate gout

Metabolic disorders:Hyperparathyroidism

HypothyroidismHypophosphatasiaHaemochromatosis

Long-term steroid therapyPossible associations:

HypertensionRenal insufficiency

AcromegalyPaget's diseaseDiabetes mellitusWilson's disease

pyrophosphatases, in which 2 mol oforthophosphate (Pi) are produced permol of PP1 cleaved. Such enzymesinclude glucose- 6-phosphatase andalkaline phosphatase, as well as more

specific inorganic pyrophosphatases.The metabolic importance of PP1

has yet to be defined. The assumptionthat intracellular concentrations of PP1are very low led Kornberg and others'61 to point out that the removal of PP,provides a means of drivingpyrophosphorylase reactions in thedirection of synthesis, essentiallyrendering them irreversible. However,observations of detectable amounts ofPPi in rat liver, for example,'8 '9question this assumption, and,moreover, question whether the

Disturbed PP1 metabolism.Physical damage to chondrocytes.Release of nucleating agents or decreased

inhibitory activity (perhaps via action of releasedproteases to destroy inhibitors). Increasedcartilage permeation by Ca"+ and PP1.

Epitaxy on apatite crystals.pH may be lowered during inflammation whichpromotes crystal transformations to moreinsoluble forms.

Epitaxy on urate crystals

Raised extracellular Ca, and/or raised PP1 due toincreased adenylate cyclase activity.

Metabolic changes in cartilageRaised PP1 due to alkaline phosphatase deficiencyFe as nucleating agent or

pyrophosphatase inhibitorSecondary hyperparathyroidism

High PP, in chronic renal failure; acidosis promotescrystal transformation to insoluble forms

Metabolic alterations in cartilageAge related

Copper as crystal nucleating agent or

pyrophosphatase inhibitor

hydrolysis reaction for PP1 is atequilibrium in vivo. An alternativepossibility may be that inorganicpyrophosphatase is a nonequilibriumenzyme and that its activity is thelimiting factor in the removal of PP1.Additional determinants of theintracellular steady stateconcentration of PP1 may be theintracellular concentration of Pi, bothas a result of the effect of Pi on theequilibrium reaction, and because it isa competitive inhibitor ofpyrophosphatases. If thepyrophosphatase reaction is not inequilibrium, the concentration of PPiwill be determined by the balancebetween the rate of formation andbreakdown of PPi. In this case, the

concentration of intracellular PPiwould be under metabolic control andcould respond to changes in the ratesof either its synthesis or degradation.Furthermore, if the intracellularconcentration of PP1 does change inresponse to different metabolicconditions, it becomes possible for thision itself to be involved in theregulation of metabolism.

IS THE PPi HYDROLYSIS

REACTION IN EQUILIBRIUM?Attempts to determine theequilibrium constant and free energychange for hydrolysis of PP, understimulated physiological conditionshave produced variable results.20 22However, the reported tissueconcentrations of PPi appear to exceedthose calculated from values found forthe equilibrium constant,'8 19 23supporting the view that the hydrolysisreaction is not in equilibrium. Usingfreeze-clamped rat tissues we haverecently demonstrated that the totalPP, content of skeletal muscle (50nmol/g) is substantially higher thanthat found in liver, kidney, heart, andlung tissues (20 nmol/g) (unpublishedobservations). Similarly, with isolatedhuman cells, it has been observed thatfibroblasts and synovial cells containless PP1 than chondrocytes and bonecells. Furthermore, the higher PP1content of chondrocytes relative tofibroblasts appears to be correlatedwith differences in the rate ofproteoglycan synthesis in these twocell types.24.26

Several studies have shown thatboth the total PP1 content and thecalculated cytoplasmic free PP1content of freeze-clamped rat livermay change quite considerably aftervarious short term or long termmetabolic manipulations-forexample, after administration ofacetate or butyrate or after 48 hours'starvation."9 21 23 Such studies haveled to the suggestion that hepaticglucose uptake and phosphorylationare regulated predominantly bychanges in the concentration of freePPi in cytoplasm,2' thereby indicatinga role for PP1 in metabolic regulation.An increase in the intracellularconcentration of PP1 in response to thestimulation of a biosynthetic pathwayproducing PP1 has also been observedin isolated human articularchondrocytes after enhancement of



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Pathogenesis ofchondrocalcinosis and pseudogout Suppl p 29

glycosaminoglycan synthesis bytreatment with xylosides.2"

Raised intracellular concentrationsof PPi have also been reported inisolated fibroblasts and lymphoblastsderived from patients of a Frenchfamily with an hereditary form ofchondrocalcinosis.12 27 Thisobservation raises the question ofwhether some of the hereditary formsof chondrocalcinosis are associatedwith overproduction of PP, in cells,due to an abnormality in one of thebiosynthetic pathways generating PP,.Reports of abnormal cartilage matrixproduction in patients from twoSwedish families with a hereditaryform of this disease might support thisview.'2 28Hence evidence appears to indicate

that the intracellular concentration ofPP, does vary with such factors as celltype, metabolic conditions, and thepresence of disease states, supportingthe hypothesis that the hydrolysisreaction is not in equilibrium and thatthe concentration of this ion is undermetabolic control.

CONTROL OF THEINTRACELLULARCONCENTRATION OF PPiFactors involved in the control of theintracellular PP, concentration haveyet to be defined. As discussed above,PPi production will be controlled bythose factors which regulatebiosynthetic pathways containing PPigenerating steps and it will benecessary to delineate the relativecontribution of the different pathwaysto the overall rate of PP, production inthe cell. PP, breakdown will becontrolled by those factors whichregulate inorganic pyrophosphateactivity.

TISSUE DISTRIBUTION ANDPROPERTIES OF INORGANICPYROPHOSPHATASEA recent study of inorganicpyrophosphatase activity in the ratshowed that the total activity variesquite considerably between tissues andthat the total hepatic activity changesduring development.2' These resultsimply that the total tissue activity ofthis enzyme is regulated, but it shouldbe noted that the activities measuredin this study under optimal conditionsin vitro do not necessarily reflect thereal activities in vivo.

Inorganic pyrophosphatase appearsto be principally located in thecytosol.a 32 Smaller amounts of theactivity occur in mitochondria.33Inorganic pyrophosphatase activityhas also been demonstrated inendoplasmic reticulum in some tissues,but this may be due to the presence of aglucose-6-phosphatase,4 35 which alsopossesses pyrophosphatase activity.

Studies of pyrophosphatase in thecytosol from various tissues (red bloodcells, polymorphonuclear leukocytes,cartilage, dental pulp, etc.) havesuggested that this enzyme is specificfor PPi31 32 ...39 has a pH optimum of7-8, requires Mg++ for activity, isstrongly inhibited by other divalentmetal cations even in the presence ofMg++-for example, Ca++, Fe", andCu'' and is also inhibited by fluorideions. Several known inhibitors ofalkaline phosphatase-for example,Pi, imidazole, and CN- have littleinhibitory effect on this enzyme andthis, together with its specificity,suggest that this inorganicpyrophosphatase activity is a functionof a discrete enzyme and is not simplya function of alkaline phosphatase.Reports of the Km for PP1 of acytosolic inorganic pyrophosphataseare in the range 11-40 mmol, whichmay imply that the enzyme is notsaturated with substrate underphysiological conditions.Mitochondrial inorganic

pyrophosphatase activity occurs in twoforms, one of which is membranebound. Both forms resemble thecytosolic activity in that they arespecific for PPi, require Mg++ foractivity, have pH optimum of 7-8 andare inhibited by divalent cations-forexample, Ca++-and by fluoride.33 4 4

The effects of Mg++ and Ca++ may beof particular relevance and we are atpresent examining the influence of theavailability of these cations on theintracellular concentration of PP, inhuman articular chondrocytes andbone cells.

Extracellular metabolism of inorganicpyrophosphate

GENERAL CONSIDERATIONSAlthough PPi occurs extracellularly inbody fluids-for example, serum,plasma, urine, saliva, and synovialfluid, and large quantities also occuradsorbed to bone mineral, there is

little information about the movementof PPi across cell membranes or aboutwhich organs make appreciablecontributions to the PP, content ofextracellular fluid. There is evidencethat this PP, is of endogenous origin;thus it is not derived direct from thediet, as dietary PP1 andpolyphosphates appear to becompletely hydrolysed to Pi within theintestinal lumen, probably by theaction of alkaline phosphatase, whichis present in the brush bordermembranes of enterocytes.42

Studies of PP, turnover have beenrestricted to the examination ofextracellular PP,. Studies using32P-labelled PPi indicate that plasmaPP, turns over extremely rapidly indogs and in man.4" In dogs thehydrolysis to Pi accounts for at least25% of the loss of PP1 from the plasmacompartment whereas urinaryexcretion accounts for only 10%.43Hence hydrolysis to Pi would appear tobe a major mechanism for the removalof PP, from the plasma compartment.32P-labelled PP1 added to whole bloodin vitro, however, is only relativelyslowly hydrolysed, implying that themajor hydrolytic enzymes are notcirculating but are located on or withincells.43

ORIGIN OF EXTRACELLULAR PPiIN ARTICULAR CARTILAGEIf CPPD crystals are first formedoutside cells rather than inside, thenthe origin of extracellular PP, andmechanisms controlling the localconcentrations of this ion becomeimportant. (Fig. 1)The PP, present in the extracellular

space of articular cartilage could eitherarise from the intracellular compart-ment or it could be synthesisedextracellularly or released from sub-chondral bone. Release of PP, fromthe intracellular compartment couldoccur by a variety of mechanisms--forexample, by a specific membrane car-rier for PP,, in conjunction with thesecretion of matrix components or fol-lowing cell damage or death.

THE RELEASE OF PP1 FROMARTICULAR CARTILAGEGeneration of PPi in vitro by cartilagefragments was first reported byHowell's group,45 who stated that PPiwas released from growth platecartilage and from articular cartilage



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INTRACELLULARSPACE

Posextr

Nucleosidetriphosphate

(NTfP) -~~~/BLoaynthetic reactionseg proteoglycan synthesis7

NUCLEOSIDE TRIPHOSPHATEP YROPHOSPHORYIASES

derived from young rabbits and frompatients with osteoarthritis but notfrom rabbit ear cartilage or fromarticular cartilage derived frommature rabbits or 'normal' humans.However, in a recent study, McCarty'sgroup,4" using a highly sensitive assayand correcting for PP1 hydrolysisduring the incubation by.the use of32P-labelled PPi, have observed release

EXTRACELLULARSPACE

cAMP

2P.

release ofproteoglycans

plus PP iin vesicles

leakage ?

cell damage ?

NUCLEOSIDE TRIPHOSPHATEPYROPHOSPHOHYDROLASE

(ectoenzyme)

+ PPI

of PPi from fragments of articularcartilage and fibrocartilage derivedfrom both young and adult rabbits andfrom fragments of 'normal' humanarticular cartilage. Extrusion of PP,may therefore be a feature common toall types and.age of cartilage.

It has been reported that, if washedmonolayers of human articularchondrocytes are incubated in

phosphate-buffered saline or mediumwithout serum, no detectable releaseof PP, from these cells occurs.Furthermore, when the concentrationof PPi in the medium is raised toaround 100 ,.tmol/l rapid hydrolysis ofPP; occurs.24 47 However, using a moresensitive assay, we have recently beenable to demonstrate the release ofsmall amounts of PP, from washedmonolayers of human articularchondrocytes incubated in mediumwithout serum. Additionally, we haveobserved that under our conditions, inwhich the extracellular PP,concentration is generally less than 1,tmol/l PP1 hydrolysis determined with32P-labelled PPi in the medium is veryslow (A. M. Caswell and others, p. 99).This difference between our resultsand those of others47 may imply thepresence of an extracellular inorganicpyrophosphatase activity with arelatively high Km for PP,.These various results suggest that

chondrocytes possess the ability torelease limited amounts of PP,. In thiscontext, it may be relevant that it hasnot been possible to show the passageof PPi across the membrane of the redcell.46 It is also of interest that, in thestudy of McCarty's group describedabove, release of PPi from rabbitcartilage fragments, but not fromhuman cartilage fragments, waspositively correlated with release ofuronic acid.46 However, this does notnecessarily establish that PP, anduronic acid are released from the celltogether since, as suggested earlier,changes in the rates of synthesis ofmatrix components could result inparallel changes in intracellular PP,.Such changes could in turn influencethe rate of release of PPi from the cellby any putative carrier mechanismsspecific for PPi. Release of PP, into theextracellular space of cartilage afterdamage or death of chondrocytes mayoccur and could explain why.patientswho have had meniscectomies have ahigher incidence of chondrocalcinosisin the operated knee than in theunoperated knee.49 The extent towhich this release mechanism occurs inundamaged cartilage or in other'normal' tissues is not known.

PRODUCTION OF PPiEXTRACELLULARLYAnother possible origin ofextracellular PP, is that it is generated

* PPi

PYROPHOSPRATASE(ectoenzyme)eg alkalinephosphatase

2PI

2Pi

Fig. 1 Pyrophosphate metabolism: a schematic representation ofpossiblesources of both intracellular and extracellular PPi. PPi introduced in theintracellular compartment may be either co-secreted with products ofintracellularbiosynthetic reactions-for example, proteoglycans-or may leak to theextracellular compartment when cells are damaged, as happens in degenerativejoint disease. Extracellular PP, may arise from the activity of the ectoenzyme,nucleoside triphosphate pyrophosphohydrolase, acting presumably on nucleosidetriphosphates 'leaked' to the extracellular compartment.



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outside the cell by membrane boundenzymes. One such enzyme is adenylatecyclase which catalyses the reaction:

ATP---3' ,5'-cyclic AMP + PP,.

This enzyme is located in cellmembranes and is activated inresponse to hormone and otheragents-for example, certain drugs.50The cyclic AMP formed is releasedinto the intracellular compartment butthe fate of PP, formed is unknown andit is not known whether any is releasedinto the extracellular fluid.Another enzyme of considerable

interest is nucleoside triphosphatepyrophosphohydrolase, whichcatalyses the reaction:NTP--NMP+ PP,, where NTP de-

notes nucleoside triphosphate andNMP nucleoside monophosphate.

In rat liver the enzyme is located inthe plasma membrane, is calciumdependent and hydrolyses bothpyrimidine and purine nucleosidetriphosphates with Kms in the Molarrange.5"-53 The enzyme has beenproposed to function either in calciumtransport52 or together with5'-nucleotidase in the salvage ofnucleoside triphosphates that leakfrom the cell.53 In rats this enzyme hasbeen shown to exhibit a wide tissuedistribution and the activity appears tovary quite considerably with tissuetype, the highest activities beingobserved in liver, small intestine, andkidney and much lower activities beingobserved in brain, thymus, andblood.52The presence of a nucleoside

triphosphate pyrophosphohydrolaseactivity in human articular cartilagehas been inferred from a recent studyof Howell's group, who demonstratedthe generation of PP, after adding 1mmol/l ATP to cartilagehomogenates.54 However, the use ofhomogenates precludes any statementabout the location of this enzymeactivity or the number of possibleactivities involved.We have been able to demonstrate

the generation of substantial amountsof PPi extracellularly after adding ATP(concentration range 6-25-400 Amol/1)to washed human articularchondrocyte monolayers incubated inmedium without serum (A. M. Caswelland others, p. 00). The effect can alsobe observed with other nucleosidetriphosphates-for example,

guanosine triphosphate, cytidinetriphosphate, and uridinetriphosphate. These preliminaryobservations suggest that presence of ahighly active nucleoside triphosphatepyrophosphohydrolase on the outsideof the plasma membrane of humanarticular chondrocytes with anapparently low Km for nucleosidetriphosphates. The function of thisactivity in chondrocytes is unclear butcould be similar to that suggestedearlier for nucleoside triphosphatepyrophosphohydrolase activities inother tissues-for example, a role incalcium transport.

It is therefore interesting that thetotal concentration of nucleotides inthe extracellular fluid of chickenepiphyseal cartilage has been reportedto range from 200 ,u mol/l in theproliferating zone to 740 molIl in thehypertrophic zone.55 Thus there is apotential mechanism for theextracellular generation of PPi, at leastin growth cartilage, since appreciableamounts of PP, could be generated ifnucleoside triphosphates comprisedeven a small fraction of the observedtotal nucleotide concentration.

It is also interesting to note that inthe recent study by Howell's groupdescribed above,54 nucleosidetriphosphate pyrophosphohydrolaseactivity was greater in articularcartilage homogenates derived frompatients with chondrocalcinosis than inthose derived from patients withosteoarthritis. One sample derivedfrom a 'normal' patient containedvirtually no activity. This apparentincrease in the activity in a situation inwhich increased amounts of PP1 occurextracellularly supports the view thatthis enzyme is important in thegeneration of PP, extracellularly inarticular cartilage. Furthermore, theremay be a role for this activity in thepathogenesis of chondrocalcinosisafter cartilage damage, since theleakage of metabolites from disruptedcells could result in the release ofnucleoside triphosphates, which couldthen serve as substrates for thegeneration of PP,.

RELEASE OF PPi FROMSUBCHONDRAL BONE

Local release of PP, from subchondralbone has been suggested as a furtherpossible source of extracellular PP, inarticular cartilage. Some polarity

would be necessary so that PP, couldreach subchondral bone through theblood supply on the metaphyseal sideand then subsequently be releaseddown a concentration gradienttowards the articular surface. Thiscould account for the observation thatcalcium pyrophosphate dihydratedeposits in the mid zone of articularcartilage, since this would be the pointat which PP1 from the subchondralbone and calcium from the joint spacewould come together.56

ALKALINE PHOSPHATASE ANDREMOVAL OF PPi FROM THEEXTRACELLULAR SPACEAs in the case of intracellular PP,metabolism, the major mechanism forremoving PPi from the extracellularcompartment is enzymatic hydrolysis.The addition of inorganicpyrophosphatase, to extracellularfluids--for example, plasma-results ina pronounced reduction in the PP1concentration and therefore thereaction:

PP 2Picannot be in equilibrium and thebreakdown of extracellular PP, mustbe limited by the activity of hydrolyticenzymes.The physiological importance of

alkaline phosphatase activity in theextracellular hydrolysis of PP; hasbeen demonstrated mainly by studiesof patients with hypophosphatasia inwhom a deficiency in alkalinephosphatase activity occurs."4 Thereis a fourfold increase in the plasma PP1concentration with a correspondingreduction in the rate constant ofhydrolysis in the removal of PP, fromthe extracellular compartment.5 4 5Such observations establish thatalkaline phosphatase does function asa pyrophosphatase in vivo and that isan ectoenzyme capable of hydrolysingsubstrates present in the extracellularcompartment. It can be calculated that-alkaline phosphatase may beresponsible for the removal of as muchas 80% of the PPi delivered to theextracellular compartment in normalindividuals.

PROPERTIES OF THE PPjHYDROLYTIC ACTIVITY OFALKALINE PHOSPHATA-SEThe properties of the hydrolyticactivity of alkaline phosphatasetowards PP1 has been studied in a



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variety of tissues-for example, liver,kidney, and small intestine,56bone6' 62 and epiphyseal cartilage andmatrix vesicles.63-' This enzyme exhibitsremarkably similar properties in awide variety of tissues. The pHoptimum of the PP, hydrolytic activitydecreases with decreasing PP1concentration, so that at physiologicalconcentrations of PPi approaches pH7- 0. Magnesium ions affect the activityin a complex manner and this effectappears to be influenced by theavailability of substrate with anoptimal activity occurring at aMg+/ PPi concentration ratio of 1: 1.This led to the suggestion thalt the truesubstrate for the enzyme is MgP2O72-and that this cannot form if PP1 ispresent in excess over Mg++, whereas ifMg++ is present in excess, MgP2O7forms, which is inhibitory. In thepresence of Mg++, Ca++ is only slightlyinhibitory but the activity is subject toinhibition by substrate and product(Pi). The Km of alkaline phosphatasefor PPi inhibition has been reported torange from 40-85 molIl but some ofthese differences may be accounted forby the use of different concentrationratios of Mg'+ to PP1.

In a recent study, Howell's group54have extracted and examined theproperties of alkaline phosphatasederived from adult human articularcartilage. Virtually no activity could beobtained from 'normal' samples butappreciable amounts of the activitywere obtained from patients withchondrocalcinosis and even higheractivities were obtained from patientswith osteoarthritis.

In articular cartilage derived frompatients with osteoarthritis orchondrocalcinosis, there appear to betwo forms of alkaline phosphatase,both of which possess hydrolytic-activity towards PP, but whereas oneform of the enzyme resembles theactivity found in other tissues, theother appears to be activated ratherthan inhibited when Mg` is in excessover PPi. It is not known, however,whether both forms of the enzyme arefound in normal adult human articularcartilage.Taken together, these studies of the

hydrolytic activity of alkalinephosphatase towards PP, demonstratethat this activity can vary in responseto a variety of physiologicalagents-for example, Mg++, Ca,- and

Pi, and further studies are needed todetermine whether such agentsregulate the breakdown ofextracellular PP, in vivo.

IS A DEFECT IN CLEARANCE OF

PPi FROM ARTICULARCARTILAGE INVOLVEDIN THE PATHOGENESIS OFCHON DROCALCINOSIS?There is little direct evidence for adefect in PP, clearance from articularcartilage in patients with idiopathicchondrocalcinosis. There have beenreports that inorganic pyrophosphataseactivity due to both alkalinephosphatase and glucose-6-phosphatase is reduced in jointfluids from patients withchondrocalcinosis.66... In otherstudies, however, no change wasobserved either in the hydrolyticactivity of alkaline phosphatasetowards PP, or in an inorganicpyrophosphatase activity, with an acidpH optimum, in joint fluids from thesepatients.70 Moreover, as noted earlier,Howell's group observed an increaserather than decrease in the PPihydrolytic activity of alkalinephosphatase in articular cartilagederived from patients withchondrocalcinosis.54

However, some of the diseaseassociations observed suggest thatthere is a defect in PP, clearancefrom articular cartilage at least in somecases of chondrocalcinosis-forexample, in hypophosphatasia.7'Similarly, the associations betweenchondrocalcinosis and hyperpara-thyroidism,7274 haemochromatosis7'and hypomagnesaemia73 couldreflect the influence of Ca++, Fe++,and Mg++ respectively on the pyro-phosphatase activity of alkaline phos-phatase.

CLINICAL CONDITIONS IN WHICHDISORDERS OF EXTRACELLULARPP1 METABOLISM OCCURMeasurements of PP, in serum,plasma, or urine in a variety of clinicalconditions have suggested thatdisorders of extracellular PP,metabolism do occur in some diseasestates, hypophosphatasia being thebest example. However, plasma andserum PPi concentrations are alsoraised in about one third of patientswith chronic renal failure, and thevalues return to normal after

haemodialysis or renal transplant.76 'An increase in the PP, content of bonehas also been noted in some patientswith chronic renal failure and it hasbeen suggested that a relationshipexists between the bone content of PP,and the extent of soft tissuecalcification in these patients.79The plasma concentration of PP, has

been reported to be raised in somecases of acromegaly and this increaseappears to be correlated with anincrease in the plasma Piconcentration.' "' Plasma PP, is alsoraised in some patients withosteomalacia due to vitamin Ddeficiency, but apparently not in someof the inherited forms of vitaminD-resistant renal tubular rickets, norin osteomalacia associated with totalparenteral nutrition,'5 (M. K. B.McGuire and others, paper presentedat 14th Annual Meeting of AmericanSociety of Nephrology, 1981).A defect in urinary PP, excretion

may contribute to formation of renalstones in some cases. In several studiesit has been reported that urinary PP1excretion is reduced in men who formstones but not women, with this effectbeing most marked in the 30-40 agegroup."-83A defect in extracellular PPi

metabolism is unlikely in osteogenesisimperfecta as we have been unable toconfirm the observation of Solomon'sgroup that the serum PPiconcentration is raised in thisdisorder."5

In this context it is important to notethat measurements of PP, in serumgive values two to four-fold higherthan in plasma. This is due to therelease during blood clotting of PP,stored in the dense granules ofplatelets.

There is little evidence for anychange in the plasma PP,concentration in either rheumatoidarthritis or osteoarthritis.'7 8 86-86

In summary, there appears to be nosystemic disorder in PP1 metabolismassociated with most cases ofchondrocalcinosis.

Physicochemical studies of calciumpyrophosphate crystal formation

Little is known about thephysicochemical conditions necessaryfor the formation of calciumpyrophosphate dihydrate (CPPD)



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crystals in articular cartilage and othersites in vivo. The problem can beconsidered from several points ofview:(a) What factors are necessary forinitiating crystal deposition? (b) Whatdetermines the interconversions ofdifferent crystal forms of CPPD? (c)What influences the formation of thecrystals, their solubility and removalfrom the joint?

INITIATION OF CRYSTALDEPOSITIONThe formation of CPPD crystals inpathological conditions may bepromoted in various ways. Firstly, theconcentrations of calcium or PPi maybe raised; the increasedconcentrations of PP, could result fromeither enhanced production ordecreased removal from the joint, as,for example, in hypophosphatasia." "712By analogy, increased plasma calciumconcentrations occur inhyperparathyroidism, althoughwhether or not the association withdeposition of CPPD crystals ismediated via raised calciumconcentrations is at present unknown.Crystal formation might also befavoured by the presence of nucleatingagents or by the removal of any neutralinhibitors of crystal formation,although currently very little is knownabout these possible mechanisms. Inorder to study some of these questionswe have devised a simple method todefine the conditions necessary forcrystal deposition in vitro.89 90The formation of crystals was

studied in simple synthetic solutionswhich mimic extracellular and synovialfluids. At physiological concentrationsof calcium (1- 5 mmol/) at pH 7- 4 andphysiological ionic strength, crystals ofcalcium pyrophosphate were found toform within three days at 370C, whenthe PPi concentration was 40 ,umol/l orhigher. In the presence of Mg++ atphysiological concentrations (0.5mmolIl), crystals formed only whenthe PP, concentration reached 175mol/l. This contrasts with theconcentrations of PP, found in normalsynovial fluids (mean 3 molIl; range 1to 4 mol) and in pseudogout fluid(mean 20 ,umolVl; range 5 to 60,umol/l). In the presence ofphysiological concentrations (1mmol/l) of inorganic phosphate(orthophosphate) the PPi

concentration required for crystalinitiation was lowered to 75 molIl. Thisreflects conditions which are theclosest to physiological that have so farbeen explored. It appears, therefore,that the formation of CPPD crystals insynovial fluid would not be favoured invivo. To explain where and whycrystals form in the body one thereforeneeds to invoke additionalmechanisms such as increased localconcentrations of either calcium orPP,, or the presence of nucleatingagents.The concept of nucleating

mechanisms leading to crystalformation is an attractive one whichrequires further study. The associationof pyrophosphate arthropathy withhaemochromatosis7" was investigatedand the possibility that iron salts mightact as nucleating agents for crystalformation was tested in the crystalgrowth system in vitro. The presenceof low concentrations of ferric salts(Fe++ at 25 umol/l) promoted crystalgrowth with the result that the amountof PPi needed for crystal formationwas reduced to one quarter of thatrequired in the absence of iron.89 It wasthought that this effect may have beendue to the colloidal nature of Fe-+ saltsin solution at neutral pH. Urate atphysiological concentrations also has asmall promoting effect on theprecipitation of calciumpyrophosphate'9 but it is doubtfulwhether this effect is potent enough toexplain the association found betweenurate gout and pyrophosphatearthropathy.

Crystal formation in vitro increasesrapidly as the pH rises through therange of 7 2 to 7-4 suggesting that invivo a small change in pH could inducecrystal formation without such largechanges in the concentration of Ca andPP, being required. These results areinteresting in relation to studies ofHowell and Pita's group,9'" who haverecorded high pH in extracellular fluidaspirates from epiphyseal cartilage bymicropuncture techniques. The pH offluid within articular rather thanepiphyseal cartilage is, however, notknown. In our studies of crystalformation in vitro a high pH promotedcrystal formation.The possible role of nucleation by

epitaxy has also been explored andhydroxyapatite crystals were tested fortheir effect in our crystal growth

experiments. At 40 4g/ml the addedcrystals raised the amount ofpyrophosphate required to initiateCPPD crystal growth, presumablyexplained by their ability to absorbpyrophosphate, thereby making itunavailable. At lower concentrations(4,g/ml) preliminary results suggestthat hydroxyapatite crystals arewithout effect, suggesting a balancebetween nucleating effects and surfaceadsorption of pyrophosphate. Furtherwork is required to clarify this point.

CRYSTAL TRANSFORMATIONSThe type of CPPD crystals foundnaturally in vivo are predominantly inthe triclinic form, although McCarty etal94 and Bywaters et al95 have reportedthe occurrence of the monoclinic form.Crystals produced in the three dayincubations under the experimentalconditions of Hearn and Russell' 90were shown by x-ray diffraction to beorthorhombic in the absence of Mg-+and amorphous in the presence ofphysiological concentration of Mg+.Longer incubations of one month ormore with added magnesium appear toallow the slow formation of crystals ofthe monoclinic type, followed by aslow transition to the triclinic form.The presence of 1 mmolI phosphateconsiderably increased the rate of thistransition, but even then failed toconvert a significant proportion ofcrystals to the triclinic form.The use of nucleating agents has so

far failed to influence the type ofcrystal formed from solution.Hydroxyapatite crystals promoteCPPD crystal formation but the typesformed are orthorhombic andmonoclinic. Similarly, addition ofpreformed CPPi crystals of these typesleads only to further growth of thosecrystals and new growth of similarcrystals, with no increase in transitionto the triclinic forms.These studies are based on

microscopical identification and awaitconfirmation by x-ray diffractiontechniques. Work from the soilchemists of the Tennessee RiverValley Authority,96 who investigatedcalcium pyrophosphates in relation totheir use as fertilisers, providesvaluable information about thepotential transformations that canoccur. A study of their results indicatesthat the monoclinic and triclinicvarieties of CPPD crystals appear to



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represent the stable end products of anumber of potential crystaltransformations. Regardless of thephysical nature of the first crystals todeposit, all crystals may thereforeconvert ultimately to monoclinic andtriclinic varieties in vivo.These studies shed light on the

mystery ofwhy the naturally occurringcrystals are usually of the triclinic andoccasionally of the monoclinic variety.

In an interesting study, Pritzker etal97 showed that the monoclinic andtriclinic crystals could be made to formwithin silica and gelatin gels in vitro.Their conditions were close to, but didnot match, the natural physiologicalstate. Thus the concentrations ofcalcium and PPi they started with wereconsiderably in excess of normal andthe pH was below 6 0.Our own current work also suggests

that these crystal conversions actuallyoccur under simulated physiologicalconditions, but often take a long time,which means that the existence ofthese specific crystal forms in vivo maysimply be an indication of the length oftime available for crystal conversionsto take place.

CRYSTAL FORMATION,SOLUBILITY AND REMOVAL FROMTHE JOINTIn cartilage CPPD crystal depositsappear initially in rims around cells93and the large deposits are associatedwith empty cartilage lacunaeindicating chondrocyte death.9" In thesynovium, in contrast, CPPD crystalsare found only in the phagocytic cellsof the synovial membrane.99Experimental studies using labelledcrystals support the concept that these

.c..ystal deposits are sequestered fromt,he synovial fluid rather than beingformed in the synovium itself.

It seems probable, therefore, that!CPPD crystals are formed initially inarticular cartilage, and appear in thesynovial fluid via a process ofsheddingrather than growth in situ of newCPPD crystals.Bone is another tissue with a

possible role in formation of CPPD.Bone lies close to the joint and the PP,concentrations might be expected tobe high due to the ability of PP, toadsorb to hydroxyapatite surfaces. It isalso possible that nucleating agents arelocated at the sites where crystalgrowth starts. The preferential

location of CPPD deposits in articularcartilage and other joint structuressuggests that there may be someabnormality of PPi metabolism,metabolite diffusion, or of nucleationat these sites. PPi concentrations areraised in the synovial fluid but not inthe plasma of patients withchondrocalcinosis and osteoarthritis,with chronically symptomaticjoints.70"6 100 101 It is uncertain whetherthis reflects local abnormalities in PP,metabolism or whether it is a result ofdissolution of CPPD within the jointtissues.As crystals are not regularly found

in synovial fluid in osteoarthropathy,but only in pseudogout, despite thereported similarity in synovial fluidconcentrations of PP, levels, it wouldappear that either the PP, is derivedfrom different sources in the twoconditions, or that specificmechanisms (such as nucleation ofcrystal growth) exist in the joints ofpatients with chondrocalcinosis whichlead to CPPD crystal formation.The high negative fixed charge

density of articular cartilage102 mayfavour retention of cations.Maroudas103 has suggested thatcartilage may show some selectivity forthe retention of calcium, which wouldlead to a rise in total calciumconcentrations within the cartilage.The free ionic calcium concentrationwould, however, be expected toremain similar to that in synovial fluid.However, in pathological conditions,bound calcium might be releasedduring degradation of proteoglycansand thereby initiate the formation ofCPPD crystals.

Finally, it is possible that the raisedPPi concentrations arise fromdissolution of CPPD crystals.However, Camerlain et al104 found thatcrystals incubated in vitro with jointfluid showed very little exchange with321PPPi. This contrasts with the rapidturnover of PP, in vivo and suggeststhat either the solubility product forcalcium pyrophosphate had alreadybeen attained or that one or morecomponents of the turnover system invivo were lacking. The problems ofsolubility are complex, however, andsynthetic crystals behave differentlyfrom natural ones-possibly becauseof proteins which coat the crystals andimpair dissolution. The phagocytosisof CPPD crystals is probably

important in provoking the acuteinflammatory reaction within thejoint. CPPD, as with other crystaltypes which can provokeinflammation, has been shown toinduce cell lysis. This may be the basisfor the findings of chondrocytelacunae in the vicinity of CPPDcrystals in articular cartilage.99 Thismay be due to the intracellulardissolution of crystals releasing largeamounts of calcium and therebypoisoning the cell.The question of why only certain

patients with CPPD crystal depositiondisease experience attacks of crystalsynovitis remains to be determined.Presumably certain conditions have tobe met before crystals will be 'shed'from the cartilage to provoke anattack. There appears to be anassociation of such attacks withmajor surgical interevention,especially where there is a significantpostoperative decrease in serumcalcium concentrations (para-thyroidectomy, major surgery inthe abdomen or thorax, etc).101 Anyabrupt fall in serum calcium will lowerthe [Ca] x [PPi] ion product in theextracellular fluid bathing the CPPDcrystals and may result in a looseningof these deposits within the lacunaedue to crystal dissolution. The suddendecrease in size of the crystals mayenhance their release from sites wherethey were hitherto tightly packed.Other mechanisms for release includeminor surface fractures induced bytrauma or wear.

We are grateful to the Arthritis andRheumatism Council for their support ofthis work. A. Caswell acknowledges thereceipt of a research fellowship from theMedical Research Council.

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