Isolation of inorganic pyrophosphate from bovine and human teeth

Archs

oral Bid.

Vol.

13, pp. 683-696,

1968.

Pergamon

Press.

Printed in Ct. Britain.

ISOLATION OF INORGANlC PYROPHOSPHATE FROM BOVINE AND HUMAN TEETH SYLVIABISAZ, R. G. G. RUSSELLand H. FLEISCH Laboratory

for Experimental Surgery, Schweizerisches Forschungsinstitut, Davos, Switzerland

Summary-Inorganic pyrophosphate has been detected in enamel and dentine from human and bovine teeth. The techniques used were ion exchange chromatography and paper chromatography with 3aP-pyrophosphate as marker, and precipitation as the manganese salt with subsequent infrared spectroscopy. During extraction of enamel, a part of the pyrophosphate was found to bind to an unidentified substance that altered its chromatographic behaviour. Using an isotope dilution technique, pyrophosphate was measured quantitatively in human teeth. Dentine contained 6.9 and enamel 1.3 mg pyrophosphate-P/g total-P. The possible origin and significance of pyrophosphate in teeth is discussed.

INORGANICpyrophosphate is a product of many biosynthetic reactions (KORNBERG, 1962). Its concentration in most tissues is low, however, probably because of the widespread presence of pyrophosphatases (SCHMIDT, 1951). Among mineralized tissues pyrophosphate is known to be present in bone, where it accounts for about 0.5 per cent of the total phosphorus (CARTIER,1957, 1959; PERKINSand WALKER, 1958). Recently it was suggested (FLEISCH,RUSSELLand STRAUMANN, 1966~) that the pyrophosphate in bone might be of physiological importance, since low concentrations of pyrophosphate can markedly inhibit both the growth (FLEISCHand NEUMAN, 1961; FLEISCHet al., 1966~) and dissolution (FLEISCH,MAERKIand RUSSELL,1966b) apatite crystals in vitro. It is also possible that pyrophosphate might have effects on the solubility of tooth mineral and thus influence the resistance of normal teeth to caries. However, although pyrophosphate has recently been found in saliva (SAWINSKIand COLE, 1965; VOGELand AMDUR, 1967), it has not to our knowledge been previously unequivocally described in teeth. While this text was in preparation, NEWESLEY (1967) reported the presence in bovine dentine and enamel of a substance which migrated in one paper chromatography system like inorganic pyrophosphate. Although suggestive, these results are not a clear-cut proof of the presence of this compound in tooth mineral. In this paper we demonstrate by means of various techniques the presence of inorganic pyrophosphate in dentine and enamel from human and bovine teeth, and report the absolute amounts present. These findings have been previously summarized in a preliminary proceedings report (FLEISCH, BISAZ and RUSSELL,1966a). 683

684

SYLVIABwz, R. G. G. Russian and H. FLEISCH MATERIALS

Teeth

The bovine teeth used for identification of pyrophosphate were freshly taken from the local abbatoir. The human teeth used for identification were sound first teeth of children. The quantitative determinations were carried out on freshly extracted adult human teeth stored at - 20°C before analysis. Chemicals All chemicals were of the highest available purity, generally from Merck, Darm-

stadt, Germany. a*P-labelled tetrasodium pyrophosphate was obtained from the Radiochemical Centre, Amersham, England, and its purity was checked by an isobutanol extraction procedure as described under Methods. The aaP-pyrophosphate was stored frozen in 1 per cent sodium carbonate at concentrations of 0.8 to 722 pgP/ml. It was stable for several months under such conditions. Pyrophosphatase

Crystalline yeast inorganic pyrophosphatase (4 x recrystallized) was a generous gift from Dr. M. Kunitz of the Rockefeller Institute, New York (KUNITZ, 1961). Zon exchange resin

Dowex (AG) 1 x 10, 100-200 mesh, chloride form, was obtained via Fluka AG, Buchs, Switzerland. METHODS

Preparation of enamel and dentine

The teeth were washed and cleaned in distilled water. They were then split longitudinally, and the enamel and dentine powders were prepared using a drill with a steel bur, run at low speed to prevent heating. The enamel and the dentine are of different colour and consistency, and a clear interface is visible between the two in the cross-sectioned tooth. Enamel was removed from the outer surface and dentine from the inner: care was taken not to drill up to the enamel-dentine interface with resulting mixing of the two minerals. A good separation was verified by the calcium and phosphate analyses. Extraction of pyrophosphate One hundred mg of powdered enamel or dentine were placed in an all-glass homo-

genizer in an ice bath and extracted 3 times with 3 ml of cold 10 per cent (w/v) trichoroacetic acid (TCA), centrifuging between extractions. During the first extraction, a tracer amount (0 ~2-1.7 pg) of aaP-pyrophosphate (specitic activity 1l-107 me/m-mole) was added. This represented an activity of about 500,000 counts/mm. The pooled TCA extracts were made up to 10 ml with water and a small aliquot (e.g. O-5 ml) was removed, diluted 1 : 10 with water and used for determination of radioactivity, calcium and phosphate. A concentrated solution of KOH was added to the remaining extract until a precipitate of calcium phosphate began to appear. This precipitate was centrifuged

PYROPHOSPHATE

IN DENTINE

AND

ENAMEL

685

down at 2°C. Measurements of aaP-pyrophosphate in the supematant showed that most of the pyrophosphate (more than 95 per cent) co-precipitated with the calcium phosphate. The precipitate was dissolved in a minimum quantity of 0 - 5 N-HCI and diluted to 20 ml with water. At this stage the extract could be stored frozen (-20°C) before further analysis. Ion exchange chromatography

Columns of anion exchange resin (Dowex 1 x 10, 100-200 mesh, chloride form) were prepared. The columns were made of Pyrex glass, internal diameter about 0 *8 cm, and contained 10 ml wet resin, giving a height of resin bed of about 20 cm. The resin was washed with at least 50 ml of 4 N-HCI and then with water, until the pH of the effluent was above 4. Five ml of the dissolved precipitates from the extracts of enamel and dentine prepared as above were poured onto the columns and washed in with water. Orthophosphate was then eluted with 100 ml 0.05 N-HCl and pyrophosphate eluted with O-5 N-HCl, 8 fractions of 2 ml being collected. To see if the phosphorus compound is eluted in the same fractions as added 3BP-pyrophosphate, the elution was also performed with 0.09 N-HCI. Sufficient 12 N-HCI was added to all fractions to bring the acidity to 0.4-0.5 N. Phosphate and radioactivity were measured in the various fractions, after hydrolysis of the pyrophosphate to orthophosphate in a boiling water bath for 30 min. Because of poor recoveries of 3eP-pyrophosphate from the enamel extracts, it was found necessary to add ethylene-diamine-tetra-acetic acid (EDTA) before chromatography. 1.4 g EDTA (disodium salt)/100 ml of solution applied to the column proved to be effective. Phosphate determination

Orthophosphate was analysed by a modification of the method of CHEN,TORIBARA and WARNER(1956). To each phosphate-containing solution, previously adjusted to 0*4-0*5 N with HCl, an equal volume of a solution containing 0.5 per cent (w/v) ammonium molybdate, 2 per cent (w/v) ascorbic acid and 1 N-HCl was added. The mixture was heated in a boiling water bath for 10 min. The tubes were cooled rapidly, and the extinction measured in a Beckman DU spectrophotometer at 820 rnp. The colour was stable for at least 24 hr. Enzymic hydrolysis

In some experiments the authenticity of the pyrophosphate was checked by incubating the extracts of enamel and of dentine with a highly purified inorganic pyrophosphatase before ion exchange chromatography. This was done in the following way: after co-precipitation of the pyrophosphate from the trichloroacetic acid extracts, the precipitate was dissolved in a minimum of 0.5 N-HCI as described above, and potassium chloride and magnesium chloride were added to give final concentrations of 0 - 1 M and 0 * 1 mM respectively, and the pH was brought to 6 - 5. The final dilution of the dissolved precipitate corresponded to 100 mg of dentine or enamel

686

SYLVIA BISAZ, R. G. G. RUSSELLand H. FLELWH

powder in 40 ml. Incubation was carried out for 12 hr at 3O”C, 50 pg of pyrophosphatase being added at zero, 3 and 10 hr. After enzymic hydrolysis the extracts were subjected to column chromatography as described above. Isolation of manganesepyrophosphate

One g of bovine dentine was repeatedly extracted in batches of 250 mg with 10 per cent TCA in a manner similar to that described above, 0.4pg of 3aP-pyrophosphate (specific activity 107 - 5 me/m-mole) being added initially. After alkalinization, the precipitate of calcium phosphate was dissolved and reprecipitated with HCl and KOH respectively four times. After each precipitation, the radioactivity in the supernatant was checked to ensure that co-precipitation of the 3aP-pyrophosphate had taken place. The last precipitate was dissolved in a few drops of 0.5 N-HCl and made to 5 -5 ml, with 0.05 M-sodium acetate buffer, the final pH being 4.15. The solution was clarified by centrifugation. The supernatant contained 82 per cent of the radioactivity added initially as 32P-pyrophosphate. Manganese chloride (0.6 ml of 10 per cent w/v) was then added to the supernatant, followed by 0 - 6 ml of acetone. Precipitation was allowed to proceed over night at 4°C. After centrifugation, the precipitate of the manganese salt was dissolved in a few drops of 0.5 N-HCI and the precipitation repeated at pH 4 - 15 by adding acetate buffer, manganese chloride and acetone as before. Infrared spectroscopy

The manganese salt taken for infrared spectroscopy was precipitated as above. The precipitate was washed 5 times with 1 per cent manganese lyophilized. A standard of manganese pyrophosphate was prepared manner from tetrasodium pyrophosphate. The manganese salts were potassium chloride pellets and examined between 4000 and 650 cm-l Elmer Model 221 infrared spectrophotometer with a sodium chloride

a third time chloride and in a similar made up in in a Perkin prism.

Paper chromatography

Paper chromatography was carried out on the dissolved precipitate of manganese pyrophosphate from dentine and on various fractions from the ion-exchange columns. These fractions were lyophilized and dissolved in a few drops of water prior to chromatography. Ascending paper chromatography was carried out after the method of EBEL (1953), using Schleicher and Schiill 2043 b paper and the following solvent: isopropanol-water (70 : 30, v/v) containing 4 g TCA and 0.3 ml 20 per cent NH,OH /lo0 ml. The spots were developed by BERG’S (1958) method, the paper first being sprayed with a mixture of ammonium molybdate (0.5 per cent w/v) and perchloric acid (1.75 per cent) in 0.05 N-HCl, then heated at 85°C for 45 min and finally sprayed with stannous chloride (0.4 per cent w/v in 1 N-H,SO,). Autoradiography

In some cases the paper chromatograms were subjected to autoradiography (Kodirex X-ray Film) before spraying with the molybdate-perchloric acid mixture.

PYROPHOSPHATE

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687

Counting technique

Phosphorus-32 was measured in a windowless methane gas-flow counter (Frieseke and Hoepfner FH 407) with a counting efficiency of 69 per cent. The samples (50 ,ul) were applied in duplicate onto aluminium planchets previously washed in acetone and sprayed with Plastic-Film 348 (K. Frey, Wimmis, BE, Switzerland). No appreciable self-absorption was detected. Background on this counter lay between 18-25 counts/min. At least 4000 counts were collected for each planchet, unless the count rates were very low, in which case 400 counts were collected. Calcium estimation

Calcium in extracts of enamel and dentine was measured using an EEL Titrator (Evans Electroselenium Limited, Halstead, Essex) with Calcichrome [cyclotris-7(I-azo-8-hydroxy-naphtalene-3, 6-disulphonic acid), British Drug Houses Ltd.] as indicator. Calcichrome was dissolved (17.5 mg/lOO ml) in 0.1 N-NaOH. For titration 1 ml of calcichrome solution was added to 2 *5 ml of 0 - 1 N-NaOH followed by 50-200 ~1 of the sample. Titration was carried out using a microburette (Agla Micrometer all-glass syringe, Burroughs Wellcome & Co., London) containing 5 mM-EDTA. The end-point was determined graphically. Isobutanol extraction procedure for rapid separation of 32P-labelled ortho-and pyrophosphate

When it was necessary to measure the amount of radioactivity due to 3aP-labelled ortho- and pyrophosphate in the presence of each other, the following procedure was used: 32P-orthophosphate was extracted into isobutanol: petrol ether (4 : 1 v/v) as phosphomolybdic acid according to the method of HALL (1963). 3aP-pyrophosphate remained in the aqueous phase during this extraction, so that a measure of the radioactivity in the organic and aqueous phases gave an estimate of the amount of 32Portho- and pyrophosphate respectively present in the original solution. RESULTS Ion exchange chromatography Dentine. The results of the ion exchange chromatography

of extracts of human dentine are shown in Figs. 1, 2 and 3. The elution of orthophosphate and other compounds was in each case complete with 100 ml of 0.05 N-HCI. It can be seen that an acid-labile, P-containing compound was eluted from the columns with either 0.5 N- (Fig. 1) or 0.09 N-HCI (Fig. 2) in the same position as added 3aP-labelled pyrophosphate. The specific radioactivity of this compound was similar in the two cases (see Table 1). The compound was no longer eluted if the dentine extracts were first incubated with yeast inorganic pyrophosphatase (Fig. 3). Similar results were also obtained with bovine dentine. Under these conditions therefore the acid labile phosphate compound extracted from dentine was indistinguishable from inorganic pyrophosphate. Enamel. The added 3aP-pyrophosphate marker exhibited somewhat unusual elution characteristics from the ion exchange columns when applied with extracts of enamel. Thus some of the marker was eluted even before the first orthophosphate fraction

688

S~VIA

BISAZ, R. G. G. RUSSELL and H. FLEBCH

during the elution with 0.05 N-HCl. During elution with 0.5 N-HCl, the remainder of the a8-P-pyrophosphate appeared sooner than it did from dentine extracts (Fig. 4). However, during elution with 0 *5 N-HCl an acid labile phosphorus-containing substance appeared in the same position as the saP-pyrophosphate marker. This compound was destroyed by incubation with yeast inorganic pyrophosphatase (Fig. 5). If EDTA was added to the extracts of enamel before chromatography (Fig. 6), the recovery of a2P-pyrophosphate during elution with 0.5 N-HCl was improved, although some 32P-pyrophosphate still appeared in the fraction normally containing only orthophosphate. The specific activity of pyrophosphate from enamel was similar in the presence

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RECOVERYOF

PYROPHOSPHATE FROMEXTRA~TSOF HUMANDENTINE IONEXCHANGE COLUMNS OF DOWEX 1 X 10

Conditions

Human dentine

Sample applied without EDTA, pyrophosphate eluted with 0.5 N-HCl Sample applied without EDTA, pyrophosphate eluted with 0.09 N-HCl

Human enamel

with yeast inorganic

Sample applied without EDTA, pyrophosphate eluted with 0.5 N-HCl Sample applied with EDTA, pyrophosphate eluted with O-5 N-HCI Sample applied with EDTA, pyrophosphate eluted with 0.09 N-HCl

ANDENAMELAPPLIED

Mean specific activity of *PP-pyrophosphate counts/min per Pgp

Recovery of apPpyrophosphate in pyrophosphate fractions from column (%I

6000

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19,400

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or absence of EDTA (Table 1) and during elution with O-5 N- (Fig. 6) or 0 -09 N-HCI (Fig. 7), indicating that a single substance, indistinguishable from pyrophosphate was eluted under all these conditions. Similar results were obtained with bovine enamel. Paper chromatography. Paper chromatography was carried out on various lyophilized fractions from the ion exchange chromatography and on the manganese pyrophosphate isolated from bovine dentine. The 0 - 5 N-HCl fractions from columns T

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PYROPHOSPHATE

IN DENTINE

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ENAMEL

run with human and bovine enamel and dentine, as well as the manganese pyrophosphate isolated from bovine dentine, gave well defined spots with the same RI as pyrophosphate standards. The radioactivity due to the 3aP-pyrophosphate was located by autoradiography and was in all cases in the same position as the spots identified by the phosphate stain. Apart from orthophosphate (Rz 0*68-O* 71) and pyrophosphate (R, O-40-O-44), other spots were only detected with human dentine. In this case a weakly staining spot with an R, of O-54-0-56 was also seen. ‘: 80 0x 70 .s z 60

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Isolation of manganese pyrophosphate from bovine dentine

Manganese pyrophosphate isolated from bovine dentine was identified by paper chromatography as described above and by infrared spectroscopy.

SYLVU B~SAZ,R. G. G. RUSVELL and H. FLEISCH

692

The isolated manganese pyrophosphate had a similar specik activity (11,600 counts/mm per pg) to the pyrophosphate found in the 0.09 N-HCl (11,820 counts/mm per pg) or 0 - 5 N-HCI (11,420 counts/min per pg) fractions from ion exchange chromatography of extracts of the same dentine powder. Infrared spectroscopy (see Fig. 8) showed similar characteristic peaks around 910 cm-l in the manganese pyrophosphate prepared from dentine and in the standard. WAV

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Properties ofpyrophosphate binding substances in enamel

The poor recoveries of SaP-pyrophosphate from ion exchange chromatography of enamel extracts in the absence of EDTA were shown to be accounted for by the appearance of pyrophosphate in the fractions normally containing only orthophosphate, or even earlier (see Fig. 4). Some studies were performed to investigate the possible reasons for this early appearance of pyrophosphate. Fractions containing the gBP-pyrophosphate that eluted before orthophosphate were collected, lyophilized and subjected to the isobutanol extraction procedure. It was found that the radioactivity was not extracted from the aqueous layer, thus excluding hydrolysis of the pyrophosphate to orthophosphate. The Sap-compound did not move from the origin during paper chromatography, showing that it did not behave as true inorganic pyrophosphate. It was therefore thought that the pyrophosphate in these fractions might be bound to some substance, perhaps of cationic nature, that altered its behaviour during ion exchange and paper chromatography. Attempts to remove this substance from extracts of bovine enamel by cation exchange chromatography on Dowex 50 before application to anion exchanger were partially successful and increased the recovery of 83P-pyrophosphate in the O-5 N-HCI fractions from 14 to 54 per cent. The addition of EDTA also resulted in a better recovery of *BP-pyrophosphate in the

______

1 2 3 4 5 Mean values SEM

Human Teeth

135 138 124 129 122 130 zt3

Dentine 185 177 180 172 172 177 12

Enamel

P/mg per g fresh weight

TABLE 2. ~~ROPHOSPHATE,TOTALPHOSPHATE

260 259 240 260 251 254 14

Dentine 371 394 361 378 346 370 i8

Enamel

Ca/mg per g fresh weight

0,787 l-130 o-970 O-850 0.690 0.885 10-076

O-272 O-248 O-232 o-204 o-211 O-233 f0*012

PyrophosphateP mg/g fresh weight Dentine Enamel

AND CALCIUMCONTENTOFENAMELANDDENTINEOFHWANTEETH

5.83 8.20 7.80 7.15 5.66 6-93 fO*50

I.47 140 1.29 l-18 l-23 l-31 f0.06

Pyrophosphate P mglgP Dentine Enamel

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694

SYLVIA BISAZ,R. G. G. RUSSELL and H. FLELWH

0.5 N-HCl fractions from bovine enamel (from 14 to 87 per cent), and from human enamel (see Table 1). The pyrophosphate compound which eluted early could be completely hydrolysed to orthophosphate by boiling in 1 N-HCl for 10 min. It was also completely ultrafiltrable through a Visking dialysis membrane. The susceptibility of the compound to hydrolysis by pyrophosphatase was similar to that of pure pyrophosphate. The capacity of the substance in enamel extracts to bind pyrophosphate was tested by the addition of ten times the original amount of unlabelled pyrophosphate to the extracts. This resulted in a reduction in the 3aP-pyrophosphate appearing in the early fraction (0.05 N-HCl) from 29 to 0.73 per cent, whereas it increased that in the later fractions (0.5 N-HCl) from 22 to 80 per cent. This experiment indicated that the pyrophosphate in its bound form can exchange with free pyrophosphate and that either the affinity of the binding substance is low or it is saturated with pyrophosphate in the enamel extracts. Because this binding substance was detected only in enamel and not in dentine, it was thought possible that it might originate from saliva. However, no such binding substance was detected in fresh human saliva. Pyrophosphate content of human dentine and enamel. The values obtained for 5 human teeth are given in Table 2. It can be seen that pyrophosphate concentration of enamel is about one-fifth that of dentine, whether expressed in terms of fresh weight or phosphate content. It will be noted that the calcium and phosphate content of enamel/g of fresh weight was higher than that of dentine. The actual values obtained agree well with those reported by others (SOGNNAES, 1961) and confirm that the separation of enamel from dentine was satisfactory. DISCUSSION

The various techniques used show that both enamel and dentine contain inorganic pyrophosphate. The precise location of this pyrophosphate, whether in mineral or matrix, cannot be stated definitely. However, we know that hydroxyapatite crystals have a high affinity for pyrophosphate in vitro (KRANE and GLIMCHER,1962; FLEISCH et al., 1966~). The pyrophosphate in enamel and dentine could therefore represent that absorbed from their respective bathing fluids, saliva and blood, both being known to contain low concentrations of pyrophosphate (FLEISCHand BISAZ, 1962; SAWINSKIand COLE, 1965; VOGEL and AMDUR, 1967). It is interesting to note that dentine and bone, in which the sizes of the crystals are thought to be similar, contain similar amounts of pyrophosphate. Enamel, in which the crystals are larger and therefore have a lower crystal surface area, contains about one-fifth the concentration present in dentine. Pyrophosphate might have important effects on the behaviour of tooth enamel and dentine in viva, particularly in determining their rate of dissolution. Indeed, in vitro hydroxapatite crystals, in equilibrium with low concentrations (2-3 PM) of pyrophosphate, such as those found in plasma, exhibit markedly reduced rates of growth and dissolution and reduced solubility (FLEISCHet al., 1966b). The effect of pyrophosphate on dissolution and solubility are markedly similar to those of fluoride. Pyrophosphate

PYROPHOSPHATE IN DENTlNEAND ENAMEL

695

may be a physiological protector against caries and procedures increasing its concentration, perhaps in saliva, may prove effective against this disease. One way of achieving this might be to alter the activity of pyrophosphatase in saliva. However, preliminary results showed that after feeding rats with fluoride, an inhibitor of pyrophosphatase, in concentrations of 10 and 50 ppm in drinking water, no difference could be found in the pyrophosphate content of their teeth, compared with normally fed rats. Another approach might be the synthesis of compounds similar to pyrophosphate and with analogous effects but resistant to pyrophosphatase. Acknowledgements-We are grateful to Dr. QUINAUXand Dr. L. RICHELLEfor performing the infrared analysis and to Dr. J. L. LAGIERfor supplying the human teeth. This work was supported by Grant AM-07266 from the National Institute of Arthritis and Metabolic Diseases, by Grant 3567 from the Schweizerischer Nationalfonds zur Fijrderung der wissenschaftlichen Forschung, by the Sandoz-Stiftung zur Forderung der medizinisch-biologischen Wissenschaften, and by the Emil-BarrellStiftung der F. Hoffmann La Roche & Co. R&m&-Du pyrophosphate

inorganique a BtCmis en evidence dam l’email et la dentine de dents humaines et bovines. Les techniques suivantes furent utilisees: chromatographie par &changes d’ions et chromatographie sur papier, en utilisant un marquage par du 32 P-pyrophosphate, et precipitation comme un se1 du manganese, suivie de spcctroscopie infra-rouge. Pendant le traitement de l’email, une partie du pyrophosphate se combine a une substance non identified, qui modifie son comportement chromatographique. En utilisant une technique de dilution isotopique, le pyrophosphate est mesure quantitativement dans les dents humaines. La dentine contient 6.9 et l’email 1.3 mg. de pyrophosphate-P/g total-P. L’origine possible et la signification du pyrophosphate des dents sont discutees. Zusammenfassung-Im Schmelz und Dentin von menschlichen und von Rinderzahnen ist anorganisches Pyrophosphat gefunden worden. Folgende Verfahren wurden benutzt : Ionenaustauschchromatographie und Papierchromatographie mit aaP-Pyrophosphat als Markierung, Prazipitation als Mangansalz mit nachfolgender InfrarotSpektroskopie. Wahrend der Schmelzextraktion wurde beobachtet, da8 ein Teil des Pyrophosphats an eine nicht identifizierte Substanz gebunden ist, die das chromatographische Verhalten verlnderte. Mit Hilfe einer Isotopenliisungstechnik wurde das Pyrophosphat in menschlichen Zahnen quantitativ bestimmt. Dentin enthielt 6,9 und Schmelz 1,3 mg Pyrophosphat-?/g Gesamtphosphor. Die mogliche Herkunft und Bedeutung des Pyrophosphats in Zlhnen wird diskutiert.

REFERENCES BERG, G. 1958. Ascending chromatography of polyphosphates. Analyt. Chem. X),213-216. CARTIER, P. 1957. Les constituants mineraux des tissus calcifies. V. Separation et identification des pyrophosphates dans le tissu osseux. Bull. Sot. Chim. biol, 39, 169-l 80. CARTIER, P. 1959. La minkralisation du cartilage ossifiableVII1. Les pyrophosphates du tissu osseux. BUN. Sot. Chim. biol. 41, 573-583. CHEN, P. S., TORIBARA, T. Y. and WARNER, H. 1956. Microdetermination of phosphorus. Adyt. Chem. 28, 1756-1758.

SYLVUBISAZ, R. G. G. RUSSELLand H. FLEIXH

6%

EBEL, J. P. 1953. Recherches sur les poly-et metaphosphates-I. Mise au point d’une methode de separation par chromatographie sur papier. Bull. Sot. chim. Fr. 20, 991-998. FLEISCH,H. and BISAZ, S. 1962. Mechanism of calcification: inhibitory role of _. pyrouhosphate. Nature. . _ Loid. 195,911. . Fm, H., BISAZ, S. and RUSSELL,R. G. G. 1966a. Isolierung von anorganischem Pyrophosphat aus Dentin und Zahnschmelz. Helv. Dhvsiol. nharmac. Acta 24. C82-C84. FLEISCH, H., MAERKI,J. and RUSSELL,R. G. G: 1966b. Effect of pyrophosphate on dissolution of hydroxyapatite and its possible importance in calcium homeostaais. Proc. Sot. exp. B&l. Med.

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