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1972 51: 323J DENT RESH. Fleisch and R.G.G. Russell

and Diphosphonates on Bones and TeethA Review of the Physiological and Pharmacological Effects of Pyrophosphate

  

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ENZYMES, BONE, ENAMEL, AND CONNECTIVE TISSUE

R. FRANK, moderator

G. CIMASONI, moderator

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A Review of the Physiological and Pharmacological Effectsof Pyrophosphate and Diphosphonates on Bones and Teeth

H. FLEISCH and R. G. G. RUSSELLDepartment of Pathophysiology, University of Berne, Berne, Switzerland, andLaboratory for Experimental Surgery, Schweizerisches Forschungsinstitut,Davos, Switzerland; and Nuffield Department of Orthopedic Surgery,University of Oxford, Oxford, England

Inorganic PyrophosphateINORGANIC PYROPHOSPHATE AND PHYSICAL

CHEMISTRY.-Inorganic pyrophosphate (PP,)at concentrations as low as 10-6 Ml caninhibit the precipitation of calcium phos-phate from solution. It also slows the con-version of amorphous calcium phosphateto a crystalline form,2'3 without inhibitingthe formation of the initial phase of amor-phous calcium phosphate.4 The formationof amorphous calcium phosphate is accel-erated by PPI.5 Hydroxyapatite crystalshave a high affinity for Pp1,6-8 and whentreated with PP,, they can no longer induceprecipitation from supersaturated solutions.9Crystals treated with PPI show a decreasedrate of dissolution in vitro.9-12 Pyrophos-phate and polyphosphates also inhibit theprecipitation and dissolution of other salts,such as calcium carbonate,'3'4 calciumoxalate,15 and strontium sulfate.16 PPiprobably inhibits heterogenous nucleationand subsequent growth of crystal nuclei ofhydroxyapatite. PP, may interfere with theconfiguration of the active sites of disso-lution.12,17

PPI AND CALCIFICATION.-Physicochemi-cal findings suggest that PP1, which is pres-ent in normal plasma (1 to 6 WM),I-8 urine(10 to 100 FtM),19-21 and saliva (0.1 to1 RM),22 may play a role in preventingcalcium salts from depositing in abnormalbody sites, such as soft tissues, the urinarytract, and the mouth. In living animals,PPI given subcutaneously can prevent thecalcification of soft tissues such as aorta23and skin,2425 when it has been induced

This study was supported by USPHS Grant AM-7266, by the Swiss National Fund No. 3567, by theProcter & Gamble Company, Cincinnati, Ohio, USA,and by the Medical Research Council of Great Britain.

324

experimentally. It is possible that normalcalcification in the body cannot take placebefore the removal of PPj.26The pyrophosphatase required to remove

PPI could be the neutral pyrophosphatasethat appears in costal cartilage at the timeof onset of calcification and disappearsthereafter.27 Alternatively, the enzyme nec-essary might be alkaline phosphatase, whichpossesses pyrophosphatase activity,28-3 andwhich has been associated with the processof calcification.32 It recently was shownthat alkaline phosphatase and pyrophos-phatase activities in epiphyseal cartilageare associated with extracellular vesicles,33which seem to be the sites at which min-eral first deposits. Furthermore, the alkalinephosphatase in bone cells seems to be lo-cated on the outer surface of the cell mem-brane,34 35 which would be an appropriatesite if one of its functions were to destroyPP1 locally.

PPI AND CALCIUM MOVEMENTS IN BONE.-Mineralized tissues such as bone,36'37dentin,38 and enamel38 contain substantialquantities of PP1. PP1 may control the ratesat which calcium and phosphate ions enterand leave the mineral phase, and may keeppart of the mineral in a noncrystalline state.If the concentrations of PP, are dependenton the local activity of pyrophosphatases,these enzymes could be important in cal-cium metabolism.26 Hormones and otheragents that act on bone could do so througheffects on pyrophosphatases. This could oc-cur through induction or repression of en-zyme synthesis, through changes in theextracellular release of enzyme, or throughchanges in enzyme activity itself. For in-stance, enzyme activity could be altered

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PYROPHOSPHATE AND DIPHOSPHONATES 325

by hormone-dependent changes in ion con-centrations. Magnesium and calcium canactivate or inhibit pyrophosphatases, de-pending on the concentrations at which theyare added39'40; orthophosphate also is aninhibitor.29 Parathyroid hormone (PTH)and calcitonin have been shown to affectpyrophasphatase content of bone4l43 andits activity in isolated cells.44

If PPi has a regulatory role, its concen-trations should be capable of rapid change.Studies with 32P-pyrophosphate in dogshave shown that the rate of metabolic turn-over of PPI is high, and that the PPI contentof plasma is replaced every two to ten min-utes.45 Most of the injected PPi is hydro-lyzed to orthophosphate. These observationswere not unexpected, because PPI is pro-duced in many biosynthetic reactions, andpyrophosphatases are found in most tissues.CELL EFFECTS OF PP,.-Apart from its

physicochemical effects, PP, could influencemineral metabolism by cellular mechanisms.Thus, PPI increases the influx of 45Ca intocells in culture,46 which suggests an effecton calcium transport. When infused intorats, PPj decreases the PTH-induced urinaryexcretion of cyclic adenosine monophos-phate (AMP), which indicates a possibleinhibitory effect on adenyl cyclase.47

PPi IN HUMAN DISEASE.-Some bone dis-eases might be caused by abnormal accu-mulation or destruction of PP, in bone. Inhypophosphatasia, an inherited bone diseasein which alkaline phosphatase is deficient,48there are increased concentrations of PP1in plasma8 and urine.49 It is possible thatthis accumulation of PPi is the cause ofthe impairment of mineralization in thebone. Plasma PPi also is increased in somepatients with renal failure,50 and in somepatients with osteomalacia caused by vita-min D deficiency.'8 Although the relation-ship between PPI in plasma and in bone isnot understood, it is possible that the min-eralization defect seen in patients withthese conditions may be related to excessiveconcentrations of PPi in extracellular fluids.Other bone diseases, such as osteopetrosisand hyperparathyroidism, are not associatedwith abnormal concentrations of PPi inplasma.'8 In osteogenesis imperfecta, con-centrations of PPI in plasma were normal,'8in spite of a report5l that serum PPi wasincreased in this disease.The amount of PPI in urine may be ir-

portant in relation to the formation of uri-nary calculi. Its concentration in urine isin the range in which inhibition of theprecipitation of calcium phosphate and cal-cium oxalate is seen in vitro.1'15,20 Al-though the concentrations of PPi in urineof people who form renal stones are notgreatly different from normal,2' the feed-ing of orthophosphate, which is consideredeffective against calcium stones,52,53 in-creases the excretion of PP1,54,55 probablyby a direct effect on the kidney.56'57 It maybe relevant that orthophosphate feedingseems of no value in patients with stonesof uric acid and magnesium ammoniumphosphate, salts whose formation is not in-hibited by PPI in vitro.58

Pyrophosphate metabolism also is dis-turbed in pseudogout (pyrophosphate ar-thropathy), a condition in which calciumpyrophosphate crystals deposit in joint cav-ities, synovial membranes, articular car-tilage, and periarticular tissues, and causearthritis.59 The concentration of PPi inplasma and urine is normal in these pa-tients,60 but the concentration in synovialfluid itself is several times greater than con-trol values.60 This condition seems, there-fore, to be caused by a local disturbancein PPi metabolism.

PPi IN DENTISTRY.-Dental calculus.-The concentration of PPI in saliva couldbe important in determining whether depo-sition of dental calculus occurs or not. Theconcentrations of PPi that we found in sa-liva,22 although much lower than those re-ported in previous studies61'62 in whichless specific techniques were used, are stillin the range in which inhibition of calciumphosphate precipitation is seen in vitro. Nowthat a specific technique for detecting PP1 insaliva is available,22 the contention that sali-vary PPI is lower than normal in patientswith dental calculus6' must be reevaluated.Although endogenous PPI in saliva mightbe a factor in the prevention of calculusdeposition under normal conditions, at-tempts to reduce supragingival calculus inman by giving PPi have not been success-ful.63 However, it is unlikely that PPI orcondensed phosphates added to dentifricesor mouthwashes will persist for a reason-able length of time in the mouth, becauseof the considerable amounts of pyrophos-phatase and polyphosphatases present insaliva and salivary bacteria. A better ap-

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326 FLEISCH AND RUSSELL

proach might be to use compounds relatedin structure to PP1 with similar physico-chemical effects on apatite formation anddissolution, but which are resistant to en-zymic hydrolysis. Such an approach usingdiphosphonate compounds recently hasgiven encouraging results.6467

Dental caries.-It is tempting to specu-late that PP1 in dentin and enamel mayinfluence the rates of formation and disso-lution of tooth mineral. Condensed phos-phates added to the diet of rats decreasedthe incidence of caries.68'69 However, PP1itself was active only weakly, and in otherstudies, actually reduced the rehardeningrate of enamel surfaces in calcifying so-lutions.70 Because pyrophosphatases in themouth are likely to reduce the potencyof topically applied PP1 or condensedphosphates, the administration of inhibitorsof these enzymes might help, and it is inter-esting that orthophosphate and fluoride,inhibitors of such enzymes, inhibit experi-mental caries. Diphosphonates have beenfound to diminish the incidence of cariesin rats.71The precise importance, if any, of the PP1

normally present in saliva and teeth mustbe evaluated by further investigation.

DiphosphonatesDIPHOSPHONATES AND PHYSICAL CHEMIS-

TRY.-Diphosphonates, which possess P-C-Pbonds in place of the P-O-P bond of pyro-phosphate, have effects similar to those ofPP1 on calcium phosphate in vitro. Theyinhibit the precipitation of calcium phos-phate from solution,4'72-74 block the trans-formation of amorphous calcium phosphateinto hydroxyapatite,4'73 bind strongly tocrystals of hydroxyapatite where they dis-place orthophosphate,6'75 and block thegrowth and dissolution of such crystals invitro.72,76'77 They inhibit, as does PP1, theprecipitation of calcium oxalate in vitro.78However, unlike PP1, they are resistant tochemical and enzymatic cleavage.79 Onlyenzymes which split monophosphates havebeen described.80DIPHOSPHONATES AND CALCIFICATION.-

These observations prompted a study ofthe effects of diphosphonates on calciummetabolism in vivo. The two that werestudied in greatest detail are disodium ethane-1-hydroxy-l,1-diphosphonate (EHDP), and

disodium dichloromethylene diphosphonate(Cl2MDP). These and several other di-phosphonates prevent the aortic and kidneycalcification that occurs in rats given largedoses of vitamin D.74 Diphosphonates areeffective whether given parenterally ororally, but PPI is effective only when givenparenterally. EHDP also is effective againstother types of soft tissue calcification, suchas the calcium deposition induced by di-hydrotachysterol in skin.81 In rats, EHDPinhibits the production of bladder calculithat are composed of calcium oxalate andcalcium phosphate, but not those composedof magnesium ammonium phosphate.78This is consistent with the ability of EHDPto inhibit the precipitation of calcium phos-phate and calcium oxalate from solutionwithout influencing the precipitation ofmagnesium ammonium phosphate in vitro.78EHDP applied topically diminishes the de-velopment of dental calculus in the rat.06465EHDP given in large doses to several

species including dog,82 cat,83 and rat,82'84'85leads to the appearance of osteoid tissuein bone, and to a histologic lesion in theepiphyseal plate that resembles classicalvitamin D-deficiency rickets. These effectsof EHDP are not seen with equivalent dosesof CI2MDP.8485 Morphological studies inrats show that with careful choice of doses,mineralization can be selectively inhibitedwhile the laying down of matrix proceedsat normal rates, although at high doses,matrix production also diminishes.85 Theeffects are reversible when the administra-tion is discontinued.82 The dose of EHDPrequired to produce these effects on bonemineralization varies according to the spe-cies, the duration of treatment, and the typeof administration. Generally, it starts atdoses of more than 1 mg P/kg daily, givenparenterally for short periods. The impair-ment of mineralization is accompanied bya reduction in calcium absorption from thegut, which is probably secondary to thedefect in mineralization, rather than thecause of it.86

Preliminary clinical trials with EHDPhave begun in man. In myositis ossificansprogressive, EHDP is able to block theprogression of calcification in most in-stances.87-90 EHDP also allows surgicalremoval of ectopic bone without recurrenceof calcification at the operation site.89

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PYROPHOSPHATE AND DIPHOSPHONATES 327

In dentistry, EHDP blocks calculus depo-sition in man when it is given in mouth-washes.64,65

DIPHOSPHONATES AND CALCIUM MOVE-MENTS IN BONE AND TEETH.-The demon-stration that diphosphonates inhibit thedissolution of apatite crystals has led toan investigation of their ability to decreasebone resorption.

In tissue culture, several diphosphonatesinhibit bone resorption induced by PTH inmouse calvaria when this resorption is as-sessed morphologically,77 and by the re-lease of 45Ca from prelabeled bone.9' Theyare effective at concentrations as low as10-7 M. C12MDP, which is a more potentinhibitor of resorption than EHDP, blocksthe PTH-induced increases of lactate, acidphosphatase, and acid pyrophosphatase.42'43These effects on the two enzymes were notproduced by calcitonin, which supports theview that calcitonin and disphosphonateswork by different mechanisms.

In vivo, EHDP and C12MDP prevent theincrease in blood calcium induced by PTHin fasting, thyroparathyroidectomized rats,which suggests that bone resorption is in-hibited.72'77 This inhibition has been dem-onstrated dramatically in young mice givenlarge doses (10 mg/kg daily) of C12MDP.This treatment produces changes in theskeleton which resembles those of the greylethal strain of osteopetrotic mice.92 Simi-larly, in the long bones of growing rats,C12MDP retards the remodeling of themetaphysis, which becomes larger anddenser.93The effects of EHDP and C12MDP on

bone resorption and other parameters ofcalcium metabolism have been assessedquantitatively in the rat with the use of 45Cakinetic techniques. Both EHDP and C12MDPcause a progressive decrease in the rate ofbone resorption (v.-). At equal doses, theeffect of C12MDP is greater than that ofEHDP.84 Bone mineralization rate, mea-sured as v0 +, is roughly parallel to thechanges in vO0, except at the highest dose ofEHDP (10 mg P/kg daily), where the dropof the mineralization is much larger, andreflects the decreased mineralization of thematrix.84 The two diphosphonates inducea slight increase in calcium retention,84 sothat the decrease in mineralization is slightlysmaller than the decrease in bone resorp-

tion for all doses of C12MDP and EHDP(except 10 mg P/kg). Therefore, total min-eral content of the bones scarcely changes.EHDP and C12MDP at doses as low as

0.01 mg P/kg daily94-96 also can inhibit thebone loss associated with immobilization inrats. The effect is present when the com-pounds are given subcutaneously or orally.Thus, doses that have effects on calciumturnover in bone in the experiments on 45Cakinetics and on immobilization osteoporosisare much lower than those that impedemineralization.

In addition to inhibition of calculus depo-sition in humans,64'65 EHDP diminishes thedissolution rate of enamel and the forma-tion of fissure and smooth surface cariesin rats.71 Also, EHDP recently has beenshown to decrease bone resorption in man.In Paget's disease, EHDP reduces urinaryhydroxyproline and plasma alkaline phos-phatase to nearly normal values.97 The ef-fects last for several weeks after the dis-continuation of the treatment, probablyreflecting the fact that EHDP is not brokendown in the body, and is liberated fromthe bone only slowly. In the rat, the half-time for retention of EHDP is betweentwo and four weeks.82'98CELL EFFECTS OF DIPHOSPHONATES.

The effects of diphosphonates on calcifica-tion and bone can be accounted for interms of their known physicochemical ef-fects on calcium phosphate in vitro. It ispossible that they may work by other mech-anisms, perhaps through effects on trans-port systems or enzymes; little informationis available about this. In bones and teeth,it may be difficult to resolve the problemof site of action because any effects seenon cell metabolism may be secondary tochanges in the mineral phase, rather thanthe cause of them. Therefore, it is necessaryto use isolated systems to study such effects.It was found recently that EHDP andC12MDP alter the rate of release of calciumfrom mitochondria in vitro.99 The effectsof diphosphonates on phosphatases andpyrophosphatases also are of interest, be-cause diphosphonates resemble pyrophos-phates, which are natural substrates forsome of these enzymes. In vitro, the diphos-phonates have no effect, or can inactivate,inhibit,100 or activate pyrophosphatases andphosphatases, according to the conditions

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328 FLEISCH AND RUSSELL

used.79 It is difficult to determine the sig-nificance of these observations in livinganimals because the concentration of sub-strate and inhibitors in loco are unknown.However, concentrations of PP1 in plasmaare normal in patients treated with EHDP.Many other modes of action are possible.The prevention by EHDP of changes asso-ciated with adjuvant arthritis in rats01' sug-gests that the known effects may not be theonly ones that are important; research onthe other effects may be rewarding.

ProspectsIn the dental field, the effects already

demonstrated on the formation of calculus,as well as on bone resorption and caries,indicate that the diphosphonates may bevaluable therapeutic tools. It will be par-ticularly important to see whether phos-phonates or similar compounds can be foundwhich have selective effects on either theformation or on the dissolution of apatite.

References1. FLEISCH, H., and NEUMAN, W.F.: Mech-

anisms of Calcification: Role of Colla-gen, Polyphosphates, and Phosphatase,Amer J Physiol 200:1296-1300, 1961.

2. FLEISCH, H.; RUSSELL, R.G.G.; BISAZ, S.;TERMINE, J.D.; and POSNER, A.S.: Influ-ence of Pyrophosphate on the Transfor-mation of Amorphous to CrystallineCalcium Phosphate, Calc Tiss Res 2:49-59, 1968.

3. TERMINE, J.D.; PECKAUSKAS, R.A.; and

POSNER, A.S.: Calcium Phosphate For-mation In Vitro: II. Effects of Environ-ment on Amorphous-Crystalline Trans-formation, Arch Biochem 140:318-325,1970.

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The Binding of Pyrophosphate and Di-phosphonates to Hydroxyapatite Crystals,submitted for publication.

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10. FLEISCH, H.; MAERKI, J.; and RUSSELL,R.G.G.: Effect of Pyrophosphate onDissolution of Hydroxyapatite and ItsPossible Importance in Calcium Homeo-stasis, Proc Soc Exp Biol 122:317-320,1966.

11. AMDUR, B.H.; SPINELLI, M.A.; MESSER,A.C.; and BRUDEVOLD, F.: Some Inter-actions of Hydroxylapatite with Polymer-ized Phosphate Compounds, abstracted,IADR Program and Abstracts of Papers,No. 118, 1963.

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15. FLEISCH, H., and BiSAz, S.: The Inhib-itory Effect of Pyrophosphate on CalciumOxalate Precipitation and Its Relation toUrolithiasis, Experientia 20:276-277, 1964.

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22. HAUSMANN, E.; BISAZ, S.; RUSSELL,R.G.G.; and FLEISCH, H.: The Concen-tration of Inorganic Pyrophosphate inHuman Saliva and Dental Calculus, ArchOral Biol 15:1389-1392, 1970.

23. SCHIBLER, D.; RUSSELL, R.G.G.; andFLEISCH, H.: Inhibition by Pyrophos-phate and Polyphosphate of Aortic Cal-cification Induced by Vitamin D3 in Rats,Clin Sci 35:363-372, 1968.

24. SCHIBLER, D., and FLEISCH, H.: Inhibi-tion of Skin Calcification (Calciphylaxis)by Polyphosphates, Experientia 22:367,1966.

25. GABBIANI, G.: Effect of Phosphates UponExperimental Skin Calcinosis, Canad JPhysiol Pharmacol 44:320-327, 1966.

26. FLEISCH, H., and RUSSELL, R.G.G.: Pyro-phosphate and Polyphosphate. Interna-tional Encyclopedia of Pharmacology andTherapeutics, Oxford: Pergamon Press,1970, pp 61-100.

27. ALCOCK, N.W., and SHILS, M.E.: Asso-ciation of Inorganic PyrophosphataseActivity with Normal Calcification ofRat Costal Cartilage In Vivo, Biochem J112:505-510, 1969.

28. FERNLEY, H.N., and BISAz, S.: Studies onAlkaline Phosphatase. Phosphorylation ofCalf-Intestinal Alkaline Phosphatase by32P-labelled Pyrophosphate, Biochem J107:279-283, 1968.

29. FERNLEY, H.N., and WALKER, P.G.:Studies on Alkaline Phosphatases: Inhi-bition by Phosphate Derivates and theSubstrate Specificity, Biochem J 104:1011-1018, 1967.

30. RUSSELL, R.G.G.; BiSAz, S.; CASEY, P.;and FLEISCH, H.: Further Studies on theRole of Pyrophosphate in MineralisedTissues and on the Function of Phos-phatases, in Proceedings of the 5th Euro-pean Symposium on Calcified Tissues,Paris: SEDES, 1968, pp 65-69.

31. EATON, R.H., and Moss, D.W.: PartialPurification and Some Properties of Hu-man Bone Alkaline Phosphatase, En-zymologia 35:31-39, 1968.

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33. ALI, S.Y.; SAJDERA, S.W.; and ANDERSON,H.C.: Isolation and Characterization ofCalcifying Matrix Vesicles From Epi-

physeal Cartilage, Proc Nat A cad Sci(USA) 67:1513-1520, 1970.

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38. BISAZ, S.; RUSSELL, R.G.G.; and FLEISCH,H.: Isolation of Inorganic PyrophosphateFrom Bovine and Human Teeth, ArchOral Biol 13:683-696, 1968.

39. SOODSMA, J., and NORDLIE, R.C.: RatHeart Pyrophosphate PhosphohydrolaseActivities: Sub-cellular Distribution, Cata-lytic Properties, and Hormonal Responses,Biochim Biophys Acta 122:510-519,1966.

40. NAYUDU, P.R.V., and MILES, P.L.: Inhi-bition of Pyrophosphatase Activity ofMouse Duodenal Alkaline Phosphataseby Magnesium Ions, Biochem J 115:29-35,1969.

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42. RUSSELL, R.G.G.; MUUHLBAUER, R.; WIL-LIAMS, D.A.; REYNOLDS, J.; MORGAN, B.;Copp, H.; and FLEISCH, H.: DifferencesBetween the Effects of Calcitonin andDiphosphonates on Bone Resorption In-duced by Parathyroid Hormone in TissueCulture and on the Osteoporosis of Im-mobilisation in Rats, 4th ParathyroidConference, Chapel Hill, North Carolina,March, 1971. Excerpta Medica, CongressSeries, in press.

43. MORGAN, D.B.; MONOD, A.; RUSSELL,R.G.G.; and FLEISCH, H.: Effect of Di-phosphonates on Enzyme Changes Inducedby Parathyroid Hormone in Tissue Cul-ture, in preparation.

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45. JUNG, A.; RUSSELL, R.G.G.; BISAZ, S.:MORGAN, D.B.; and FLEISCH, H.: Fateof Intravenously Injected Pyrophosphate-32p in Dogs, Amer J Physiol 218:1757-1764, 1970.

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47. RASMUSSEN, H.; FEINBLATT, J.; NAGATA,N.; and DELONG, A.: Regulation of BoneCell Function, in BARZEL, U.S. (ed):Osteoporosis, New York: Grune andStratton, 1970, pp 187-198.

48. FRASER, D.: Hypophosphatasia, Amer JMed 22:730-746, 1957.

49. RUSSELL, R.G.G.: Excretion of Inor-ganic Pyrophosphate in Hypophosphatasia,Lancet 11:461-464, 1965.

50. RUSSELL, R.G.G.; BIsAZ, S.; and FLEISCH,H.: Pyrophosphate and Diphosphonates inCalcium Metabolism and Their PossibleRole in Renal Failure, Arch Int Med 124:571-577, 1969.

51. SOLOMONS, C.C., and STYNER, J.: Osteo-genesis Imperfecta: Effect of MagnesiumAdministration on Pyrophosphate Me-tabolism, Calc Tiss Res 3:318-326, 1969.

52. HOWARD, J.E.: Urinary Stone, CanadMed Assn 1 86:1001-1007, 1962.

53. THOMAS, W.C., and MILLER, G.H.: Inor-ganic Phosphates in the Treatment ofRenal Calculi, Modern Treatment 4:494-504, 1967.

54. FLEISCH, H.; BISAZ, S.; and CARE, A.D.:Effect of Orthophosphate on Urinary Py-rophosphate Excretion and the Preventionof Urolithiasis, Lancet 1:1065-1067, 1964.

55. RUSSELL, R.G.G.; EDWARDS, N.A.; andHODGKINSON, A.: Urinary Pyrophosphateand Urolithiasis, Lancet 1:1446, 1964.

56. RUSSELL, R.G.G., and FLEISCH, H.: Pyro-phosphate and Stone Formation, in HODG-KINSON, A., and NORDIN, B.E.C. (eds):Renal Stone Research Symposium, Lon-don: J.&A. Churchill Ltd., 1969, pp165-180.

57. THIELMANN, K.; SCHULZE, M.; SIEGMUND,S.; and HOEPPENER, H.: Biochemischeund klinische Aspekte des Pyrophos-phatstoffwechsels IL., Acta Biol MedGerman 22:687-698, 1969.

58. FLEISCH, H.; BISAZ, S.; and RUSSELL,R.G.G.: Influence of Pyrophosphate onthe Crystallisation of Uric Acid andMagnesium Ammonium Phosphate andIts Implications in Phosphate Therapy forUrolithiasis, Urol Int 22:483-491, 1967.

59. MCCARTY, D.: Pseudogout: ArticularChondrocalcinosis, in HOLLANDER, J.L.(ed): Arthritis and Allied Conditions, 6thed, Philadelphia: W.B. Saunders, 1966.

60. RUSSELL, R.G.G.; BISAZ, S.; FLEISCH, H.;

CURREY, H.L.F.; RUBINSTEIN, H.M.;DIETZ, A.A.; BOUSSINE, I.; GABAY, R.;MICHELI, A.; and FALLET, G.: InorganicPyrophosphate in the Plasma, Urine andSynovial Fluid of Patients With Pyro-phosphate Arthropathy (Chondrocalcino-sis or Pseudogout), Lancet 11:899-902,1970.

61. VOGEL, J.J., and AMDUR, B.H.: InorganicPyrophosphate in Parotid Saliva and ItsRelation to Calculus Formation, ArchOral Biol 12:159-163, 1967.

62. SAWINSKI, V.J., and COLE, D.F.: Phos-phate Concentrations of Sterile HumanParotid Saliva and Its Relationship toDental Disorders, J Dent Res 44:827,1965.

63. KINOSHITA, S., and MUEHLEMANN, H.R.:Effect of Sodium Ortho- and Pyrophos-phate on Supragingival Calculus, HelvOdont Acta 10:46-48, 1966.

64. MUEHLEMANN, H.R.; BOWLES, D.;SCHATT, A.; and BERNIMOULIN, J.P.: Ef-fect of Diphosphonates on Human Su-pragingival Calculus, Helv Odont Acta14:31-33, 1970.

65. FRANCIS, M.D., and BRINER, W.W.: TheEffect of Phosphonates on Dental Enam-el In Vitro and on Calculus FormationIn Vivo, in press.

66. STURZENBERGER, O.P.; SWANCAR, J.R.; andREITER, G.: Reduction of Dental Calculusin Humans Through the Use of a Denti-frice Containing a Crystal Growth Inhib-itor, J Periodont 42:416-418, 1971.

67. MCCUNE, H.W., and TUCKER, N.B.: USPatent No. 3,488,419, January 6, 1970.

68. HARRIS, R.S.; DAS, S.K.; and NIZEL, A.E.:Cariostatic Effect of Three Types of Phos-phates When Fed Singly or in Combina-tions in the Diets of Rats, J Dent Res44:549-553, 1965.

69. KOENIG, K.G.; MARTHALER, Th.M.; andMUEHLEMANN, H.R.: Effects of SomePhosphates in a Short-Period Rat CariesExperiment, Arch Oral Biol 3:258-270,1961.

70. FEAGIN, F.F.; WALKER, A.A.; and PIGMAN,W.: Evaluation of the Calcifying Charac-teristics of Biological Fluids and In-hibitors of Calcification, Calc Tiss Res4:231-244, 1969.

71. REGOLATI, B., and MUEHLEMANN, H.R.:Effect of Diphosphonate and Fluoride onCaries, Fluorine Content and Dissolutionof Rat Molars, Helv Odont Acta 14:37-42, 1970.

72. FLEISCH, H.; RUSSELL, R.G.G.; BISAZ, S.;CASEY, P.A.; and MtIHLBAUER, R.C.: TheInfluence of Pyrophosphate Analogues(Diphosphonates) on the Precipitationand Dissolution of Calcium Phosphate

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In Vitro and In Vivo, Calc Tiss Res 2supply) :10-10A, 1968.

73. FRANCIS, M.D.; RUSSELL, R.G.G.; andFLEISCH, H.: Diphosphonates Inhibit For-mation of Calcium Phosphate Crystals InVitro and Pathological Calcification InVivo, Science 165:1264-1266, 1969.

74. FLEISCH, H.; RUSSELL, R.G.G.; BiSAz, S.;MUHLBAUER, R.C.; and WILLIAMS, D.A.:The Inhibitory Effect of Phosphonates onthe Formation of Calcium PhosphateCrystals In Vitro and on Aortic andKidney Calcification In Vivo, Europ JClin Invest 1:12-18, 1970.

75. JUNG, A.; BISAZ, S.; and FLEISCH, H.:Influence of Pyrophosphate on the Ex-change of 45Ca and 32P-phosphate withHydroxyapatite Crystals, submitted forpublication.

76. FLEISCH, H.; RUSSELL, R.G.G.; andFRANCIS, M.D.: Diphosphonates InhibitHydroxyapatite Dissolution In Vitro andBone Resorption in Tissue Culture andIn Vivo, Science 165:1262-1264, 1969.

77. RUSSELL, R.G.G.; MQHLBAUER, R.C.;BISAZ, S.; WILLIAMS, D.A.; and FLEISCH,H.: The Influence of Pyrophosphate,Condensed Phosphates, Phosphonates andOther Phosphate Compounds on the Dis-solution of Hydroxyapatite In Vitro andon Bone Resorption Induced by Para-thyroid Hormone in Tissue Culture andin Thyroparathyroidectomised Rats, CalcTiss Res 6:183-196, 1970.

78. FRASER, D.; RUSSELL, R.G.G.; POHLER, O.;ROBERTSON, W.G.; and FLEISCH, H.: TheInfluence of Ethane-l-hydroxy-1,l-diphos-phonate (EHDP) on the Development ofUrinary Stones in Rats, Clin Sci, in press.

79. RUSSELL, R.G.G.; PRESTON, C.; andFLEISCH, H.: Effects of Phosphonates onPhosphatases and Pyrophosphatases, inpreparation.

80. LA NAUZE, J.M.; ROSENBERG, H.; andSHAw, D.C.: The Enzymatic Cleavage ofthe Carbon-Phosphorus Bond: Purifica-tion and Properties of Phosphonatase,Biochim Biophys Acta 212:332-350, 1970.

81. CASEY, P.A.; RUSSELL, R.G.G.; andFLEISCH, H.: Effect of Sodium Etidronate,Polyphloretin Phosphate, and Polyoes-tradiol Phosphate on Soft Tissue Calcifi-cation, Experientia, in press.

82. KING, W.R.; FRANCIS, M.D.; andMICHAEL, W.R.: Effect of DisodiumEthane-1-hydroxy- 1, l-diphosphonate onBone Formation in Animals, Clin Orthop78:251-270, 1971.

83. JOWSEY, J.; HOLLEY, K.E.; and LINMAN,J.W.: The Effect of Sodium Etidronatein Adult Cats, J Lab Clin Med 76:126-133, 1970.

84. GASSER, A.B.; MORGAN, D.B.; FLEISCH,H.; and RICHELLE, L.J.: The Influence ofTwo Diphosphonates on Calcium Metabo-lism in the Rat, Clin Sci, in press.

85. RUSSELL, R.G.G.; THORNTON, T.; CASEY,P.A.; MUHLBAUER, R.C.; KISLIG, A.;FLEISCH, H.; WILLIAMS, D.A.; andSCHENK, R.: Effect of Diphosphonates andCalcitonin on the Chemistry and Quanti-tative Histology of Rat Bone, in prepara-tion.

86. MORGAN, D.B.; BONJOUR, J.-Ph.; GASSER,A.B.; O'BRIEN, K.; and FLEISCH, H.A.:The Influence of a Diphosphonate on theIntestinal Absorption of Calcium, IsraelJ Med Sci 7:384-386, 1971.

87. BASSETT, C.A.L.; DONATH, A.; MACAGNO,F.; PREISIG, R.; FLEISCH, H.; and FRANCIS,M.D.: Diphosphonates in the Treatmentof Myositis Ossificans, Lancet H:845, 1969.

88. WEISS, I.W.; FISHER, L.; and PHANG, J.M.:Diphosphonate Therapy in a patient withMyositis Ossificans Progressiva, Ann IntMed 74:933-936, 1971.

89. RUSSELL, R.G.G.; SMITH, R.; BISHOP, M.;PRICE, D.; and SQUIRE, C.M.: MYOSitiSOssificans Progressiva Treated With aDiphosphonate, in press.

90. DONATH, A., and FLEISCH, H.: Unpub-lished results.

91. REYNOLDS, J.J.; MINKIN, C.; SPYCHER,D.; MORGAN, D.B.; and FLEISCH, H.:Effects of Diphosphonates on Bone Re-sorption in Tissue Culture, in preparation.

92. REYNOLDS, J.J.; MURPHY, H.; MUHLBAUER,R.C.; MORGAN, D.B.; and FLEISCH, H.:Production of Experimental Osteopetrosisin Mice Treated With Dichloromethylene-Diphosphonate, in preparation.

93. SCHENK, R.; RUSSELL, R.G.G.; andFLEISCH, H.: The Effect of Diphospho-nates on Metaphysical Remodelling inRats, in preparation.

94. FLEISCH, H.; RUSSELL, R.G.G.; SIMPSON,B.; and MUHLBAUER, R.C.: Prevention bya Diphosphonate of Immobilisation "Os-teoporosis" in Rats, Nature 223:211-212,1969.

95. MiUHLBAUER, R.C.; RUSSELL, R.G.G.;WILLIAMS, D.A.; and FLEISCH, H.: TheEffects of Diphosphonates on "Immobili-sation Osteoporosis" in Rats, Europ JClin Invest 1:336-344, 1971.

96. MICHAEL, W.R.; KING, W.R.; and FRANCIS,M.D.: Effectiveness of Diphosphonatesin Preventing Osteoporosis of Disuse inthe Rat, Clin Orthop 78:271-276, 1971.

97. SMITH, R.; BISHOP, M.; and RUSSELL,R.G.G.: Diphosphonates and Paget'sDisease of Bone, Lancet 1:945-947, 1971.

98. MICHAEL, W.R., and KING, W.R.: Metab-olism of Disodium Ethane-l-hydroxyl-l,

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332 FLEISCH AND RUSSELL

1-diphosphonate in the Rat, Rabbit, Dogand Monkey, in press.

99. SALLIS, J.; CUMMING, D.; and FLEISCH,H.: The Effect of Diphosphonates onMitochondrial Transport of Calcium, inpreparation.

100. W6LTGENS, J.H.M.; BONTING, S.L.; andBIJVOET, O.L.M.: Influence of SodiumEthane-l-hydroxy-l,1-diphosphonate and

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Mg2+ on the Inorganic Pyrophosphatasein Calcifying Hamster Molars, Israel JMed Sci 7:406, 1971.

101. FRANCIS, M.D., and FLORA, L.: TheEffects of Disodium Ethane-l-hydroxy-l,1-diphosphonate on Adjuvant-InducedArthritis in Rats, Israel J Med Sci 7:502-503, 1971.

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