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The concentration of inorganic pyrophosphate in human saliva and dental calculus
Archs oral Biol. Vol. IS, pp. 1389-1392, 1970. Pergamon Press. Printed in Great Britain.
THE CONCENTRATION OF INORGANIC PYROPHOSPHATE IN HUMAN SALIVA AND DENTAL CALCULUS E. HAUSMANN,S. BISAZ,R. G. G. RUSSELLand H. FLEISCH Pathophysiological Institute, University of Bern, Bern, Switzerland; Department of Oral Biology, School of Dentistry, State University of New York at Buffalo, Buffalo, New York 14226, U.S.A.; Laboratory for Experimental Surgery Davos, Switzerland INORGANIC pyrophosphate (PP,) is known to inhibit the precipitation and the dissolu: tion of calcium phosphate in vitro (FLEISCHand NEUMAN,1961; FLEISCH,RUSSELLand STRAUMANN, 1966). In view of these findings, the concentration of PPi in saliva and dental calculus is of considerable interest. VOGELand AMDUR(1967) measured PP, in stimulated parotid saliva and reported that the concentrations were lower in people with heavy calculus deposits. However, their analytical technique was of doubtful specificity. In the present study we have examined human saliva and dental calculus for their content of PPi using a sensitive analytical technique developed for the measurement of PP, in biological materials (BISAZ, RUSSELLand FLEISCH, 1968; RUSSELL,et al., in 1970). Our results indicate that the concentrations of PP, are much lower than previously thought. Samples of saliva (25 ml) were collected simultaneously from both parotid ducts using Carlson-Crittenden cups, modified by UNEMOTO(1961), directly into a chilled vessel containing, prior to collection, 32P-labelled pyrophosphate, [32P]PPi, from the Radiochemical Centre, Amersham, England and, ethylene diamine tetra acetic acid (EDTA). [32P]PP, was added as a label to follow loss of PPI during the collection and the analytical procedure (BISAZ, RUSSELLand FLEISCH, 1968) and to calculate the PPi concentration using the isotope dilution principle. EDTA was added to prevent enzymatic hydrolysis. The collection of 25 ml of saliva took between 30 min to 1 hr. Flow was stimulated by placing drops of O-2 per cent citric acid, twice per min, on the lateral borders of the tongue. “Resting” whole saliva was collected from normal laboratory staff. They expectorated 25 ml into a chilled vessel containing [32P]PPi and EDTA as for the parotid saliva specimens. The samples were immediately centrifuged at 4°C for 20 min at 15,000 rpm to remove salivary sediment. Prior to the removal of calculus, the teeth were scaled “gently” to remove soft plaque. Material designated as calculus was firmly attached to tooth surfaces requiring iirm strokes with sharp scalers to be removed. Only supragingival calculus was collected. Care was taken to avoid bleeding. Calculus (5-15 mg wet wt.) was analysed as described previously for dentine (BISAZ, RUSSELLand FLEISCH,1968). The determination of PP, in saliva was essentially the same as described in detail 1389
1390
E. HAWMANN, S. BISAZ, R. G. G.
RUSSELL ANDH. FLEISX
elsewhere for plasma (RUSSELL,et al. 1970). The essential features for the purification are the following in the order indicated : ultrafiltration, co-precipitation with calcium phosphate, dissolution with hydrochloric acid, treatment with Dowex 50 cation exchanger and chromatography on Dowex 1 anion exchanger. Fractions in the elution volume of PPi were hydrolysed by boiling and the PP, determined as Pi (CHEN, TORIBARAand WARNER,1956). In order to determine the specificity of the method, some samples were incubated with inorganic pyrophosphatase (KUNITZ, 1961) before analysis. One sample each of whole saliva, parotid saliva and calculus was divided into two parts. Both parts of each sample of saliva were collected in vessels containing [32P]PPi but in these specific cases only half of the sample was mixed with EDTA since EDTA would inhibit the pyrophosphatase. The EDTA-free half of each saliva sample and of the calculus extract were treated before the anion exchange step with inorganic pyrophosphatase (crystalline, 4 x recrystallised from Dr. M. Kunitz of the Rockefeller Institute, New York). The results are shown in Tables 1 and 2. TABLE1. -l-HESPECZFICXTY OF THE
MEASUFtEMENT OF INORGANIC PYROPHOSPHATE [PP,] IN WHOLE SALIVA, PAROTID SALIVA, AND DENTAL CALCULUS*
No PPase
pg P (A)
32P (cpm)
After PPase
pg P (B)
specScity (%)
32P @pm)
A -B A
Whole saliva
l-061
30,925
0.075
0
94
calcuhls
O-381
16,820
0.016
168
95
Parotid saliva
O-198
26,780
0.135
100
32
*The elution without in these
apparent pyrophosphate content and 3zP content of the eluate fractions corresponding to the volume of inorganic pyrophosphate after ion exchange chromatography of samples with and prior treatment with inorganic pyrophosphatase. Phosphate and radioactivity were measured fractions after hydrolysis of the PP1 to Pt in a boiling water bath for 30 min.
Comparison of the amount of [32P]PPi in the elution volume of pyrophosphate from aliquots of the same sample, one of which was incubated with pyrophosphatase prior to chromatography, indicates that in each instance at least 99 per cent of [32P]PPi had been hydrolysed by the enzyme. A similar comparison of the amount of unlabelled phosphate measured as Pi, after hydrolysis in a boiling water bath, indicates less hydrolysis by pyrophosphatase of unlabelled phosphate than labelled, pure [32P]PP,. This difference reflects some contamination of the PPi-containing elution volume by other unidentified phosphates. Table 1 suggests that the technique is at least
THECONCENTRATION OFINORGANIC PYROPHoSPHATB JNHUMANSALIVAAND DENTAL CALCULUS
1391
94 per cent specific for PP, in whole saliva and calculus but only 32 per cent for parotid saliva. Table 2 shows the range of values obtained in a small number of normal persons for the PPi and Pi concentration of whole saliva, parotid saliva and dental calculus. For parotid saliva the amount of phosphate in the PP, elution volume is at the limit of sensitivity for the chemical technique for inorganic phosphate. The results do nevertheless indicate a maximum value for the concentration of inorganic PP, in parotid saliva. TABLE~.PP, ANDP,
INNORMALHIJMANSALIVAAND
DENTALCALCULUS
Whole Saliva
ws PP per mg PI
8 N-M
PI’ (PM) Mean
Range
Mean
Range
0.92
0.63-1.20
4.7
4.2-5.2
0*0!3&49
3.8
3-O-4.6
Parotid saliva
$601
0.26
calculus
(20%)
-
-
-
-
Mean
Range
4.4
2.5-6.3
The concentration of PPi measured in stimulated parotid saliva is about one thousand times less than that obtained by VOGEL and AMDUR (1967). This difference is in all probability due to a difference in the specificity of the pyrophosphate method. Their method relies on colour differences at two different times after addition of acid molybdate, whereas ours includes steps to separate contaminating substances. Our results are more in agreement with those of JENKINS, FERGUSON and EDGAR (1967) and JENKINS and EDGAR (1970). It is unlikely that the different results are due to differences in flow rate. DAWES (1969) showed that the orthophosphate concentration was markedly dependent on flow rate and the orthophosphate concentrations obtained by us and by VOGEL and AMDUR (1967) are very similar. Our results suggest a higher concentration of PP, in whole saliva than in parotid saliva. Whether this difference is due to different flow rate conditions used or to a higher concentration of PP, in submaxillary saliva or to a contribution by oral bacteria cannot be answered by our data to date. It is interesting that the concentration of PP, in resting saliva is high enough for it to have a role in the regulation of mineralization of dental plaque (FLEISCH and RUSSELL, 1970). Our results give no clue concerning the origin of PPi in calculus. It may be derived from saliva, gingival crevice fluid, blood or bacterial metabolism within plaque or from combinations of any one of these potential sources.
1392
E. HAUSMANN, S. BI~AZ,R. G. G. RUWZLL AND H. FLEISCH
Acknowledgements-E.H. is grateful to Professor HERBERTFLEISCHfor the opportunity to work in his Institute and to Procter & Gamble Co. for a travel grant. This work was supported by United States Public Health Service Grants, DE-01932-06 and AM-07266, by Grant 4564 from the Schweizerischer National fonds zur FBrderung der wissenschaftlichen Forschung. We are grateful to Miss P. BRONZ for skilled technical help. REFERENCES BISAZ, S., RUSSELL,R. G. G. and FLEISCH,H. 1968. Isolation of inorganic pyrophosphate from bovine and human teeth. Archs oral Biol. 13,683-696. CHEN, P. S., TORIBARA,T. Y. and WARNER, H. 1956. Microdetermination of phosphorus. Anal. Chem. 28, 1756-1758. DAWES, C. 1969. The effect of flow rate and duration of stimulation on the concentrations of protein and the main electrolytes in human parotid saliva. Archs oral Biol. 14,277-294. FLEISCH,H. and NEUMAN,W. F. 1961. Mechanisms of calicification: role of collagen poiyphosphates, and phosphatase. Am. J. Physiol. 200, 1296-1300. FLEISCH, H. and RUSSELL,R. G. G. 1970. Pyrophosphate and polyphosphate. In: The fnternational Encyclopedia of Pharmacology and Therapeutics, Section 51 (edited by PETERS,G. and RADOUCOTHOMAS,C.) Pergamon Press, Oxford. Chap. 3. In press. FLEISCH, H., RUSSELL,R. G. G. -and STRAUMANN,F. 1966. Effect of pyrophosphate on hydroxyapatite and its implications in calcium homeostasis. Nature, Land. 212,901-903. JENKINS,G. N., FERGUSON,D. B. and EDGAR, W. M. 1967. Fluoride and the metabolism of salivary bacteria. Helv. odont. Acta 11, 2-10. JENKINS,G. N. and EDGAR, W. M., 1970. Pyrophosphate in saliva and plaque. Abstract No. 232, I.A.D.R. 48th meeting. KUNITZ, M. 1961. An improved method for isolation of crystalline pyrophosphatase from bakers yeast. Archs biochem. Biophys. 92,210-272. RUSSELL,R. G. G., Brs~z, S., DONATH,A. and FLEISCH,H. 1970. Inorganic pyrophosphate in plasma in normal persons and in patients with hypophosphatasia, osteogenesis imperfecta and other disorders of bone. J. clin. Invest. In press. UNEMOTO,Y. 1961. Germ-free human parotid saliva. J. Osaka odont. Sot. 14, 14-21. VOGEL, J. J. and AMDUR, B. H. 1967. Inorganic pyrophosphate in parotid saliva and its relation to calculus formation. Archs oral Biol. 12, 159-163.
THE CONCENTRATION OF INORGANIC PYROPHOSPHATE IN HUMAN SALIVA AND DENTAL CALCULUS E. HAUSMANN,S. BISAZ,R. G. G. RUSSELLand H. FLEISCH Pathophysiological Institute, University of Bern, Bern, Switzerland; Department of Oral Biology, School of Dentistry, State University of New York at Buffalo, Buffalo, New York 14226, U.S.A.; Laboratory for Experimental Surgery Davos, Switzerland INORGANIC pyrophosphate (PP,) is known to inhibit the precipitation and the dissolu: tion of calcium phosphate in vitro (FLEISCHand NEUMAN,1961; FLEISCH,RUSSELLand STRAUMANN, 1966). In view of these findings, the concentration of PPi in saliva and dental calculus is of considerable interest. VOGELand AMDUR(1967) measured PP, in stimulated parotid saliva and reported that the concentrations were lower in people with heavy calculus deposits. However, their analytical technique was of doubtful specificity. In the present study we have examined human saliva and dental calculus for their content of PPi using a sensitive analytical technique developed for the measurement of PP, in biological materials (BISAZ, RUSSELLand FLEISCH, 1968; RUSSELL,et al., in 1970). Our results indicate that the concentrations of PP, are much lower than previously thought. Samples of saliva (25 ml) were collected simultaneously from both parotid ducts using Carlson-Crittenden cups, modified by UNEMOTO(1961), directly into a chilled vessel containing, prior to collection, 32P-labelled pyrophosphate, [32P]PPi, from the Radiochemical Centre, Amersham, England and, ethylene diamine tetra acetic acid (EDTA). [32P]PP, was added as a label to follow loss of PPI during the collection and the analytical procedure (BISAZ, RUSSELLand FLEISCH, 1968) and to calculate the PPi concentration using the isotope dilution principle. EDTA was added to prevent enzymatic hydrolysis. The collection of 25 ml of saliva took between 30 min to 1 hr. Flow was stimulated by placing drops of O-2 per cent citric acid, twice per min, on the lateral borders of the tongue. “Resting” whole saliva was collected from normal laboratory staff. They expectorated 25 ml into a chilled vessel containing [32P]PPi and EDTA as for the parotid saliva specimens. The samples were immediately centrifuged at 4°C for 20 min at 15,000 rpm to remove salivary sediment. Prior to the removal of calculus, the teeth were scaled “gently” to remove soft plaque. Material designated as calculus was firmly attached to tooth surfaces requiring iirm strokes with sharp scalers to be removed. Only supragingival calculus was collected. Care was taken to avoid bleeding. Calculus (5-15 mg wet wt.) was analysed as described previously for dentine (BISAZ, RUSSELLand FLEISCH,1968). The determination of PP, in saliva was essentially the same as described in detail 1389
1390
E. HAWMANN, S. BISAZ, R. G. G.
RUSSELL ANDH. FLEISX
elsewhere for plasma (RUSSELL,et al. 1970). The essential features for the purification are the following in the order indicated : ultrafiltration, co-precipitation with calcium phosphate, dissolution with hydrochloric acid, treatment with Dowex 50 cation exchanger and chromatography on Dowex 1 anion exchanger. Fractions in the elution volume of PPi were hydrolysed by boiling and the PP, determined as Pi (CHEN, TORIBARAand WARNER,1956). In order to determine the specificity of the method, some samples were incubated with inorganic pyrophosphatase (KUNITZ, 1961) before analysis. One sample each of whole saliva, parotid saliva and calculus was divided into two parts. Both parts of each sample of saliva were collected in vessels containing [32P]PPi but in these specific cases only half of the sample was mixed with EDTA since EDTA would inhibit the pyrophosphatase. The EDTA-free half of each saliva sample and of the calculus extract were treated before the anion exchange step with inorganic pyrophosphatase (crystalline, 4 x recrystallised from Dr. M. Kunitz of the Rockefeller Institute, New York). The results are shown in Tables 1 and 2. TABLE1. -l-HESPECZFICXTY OF THE
MEASUFtEMENT OF INORGANIC PYROPHOSPHATE [PP,] IN WHOLE SALIVA, PAROTID SALIVA, AND DENTAL CALCULUS*
No PPase
pg P (A)
32P (cpm)
After PPase
pg P (B)
specScity (%)
32P @pm)
A -B A
Whole saliva
l-061
30,925
0.075
0
94
calcuhls
O-381
16,820
0.016
168
95
Parotid saliva
O-198
26,780
0.135
100
32
*The elution without in these
apparent pyrophosphate content and 3zP content of the eluate fractions corresponding to the volume of inorganic pyrophosphate after ion exchange chromatography of samples with and prior treatment with inorganic pyrophosphatase. Phosphate and radioactivity were measured fractions after hydrolysis of the PP1 to Pt in a boiling water bath for 30 min.
Comparison of the amount of [32P]PPi in the elution volume of pyrophosphate from aliquots of the same sample, one of which was incubated with pyrophosphatase prior to chromatography, indicates that in each instance at least 99 per cent of [32P]PPi had been hydrolysed by the enzyme. A similar comparison of the amount of unlabelled phosphate measured as Pi, after hydrolysis in a boiling water bath, indicates less hydrolysis by pyrophosphatase of unlabelled phosphate than labelled, pure [32P]PP,. This difference reflects some contamination of the PPi-containing elution volume by other unidentified phosphates. Table 1 suggests that the technique is at least
THECONCENTRATION OFINORGANIC PYROPHoSPHATB JNHUMANSALIVAAND DENTAL CALCULUS
1391
94 per cent specific for PP, in whole saliva and calculus but only 32 per cent for parotid saliva. Table 2 shows the range of values obtained in a small number of normal persons for the PPi and Pi concentration of whole saliva, parotid saliva and dental calculus. For parotid saliva the amount of phosphate in the PP, elution volume is at the limit of sensitivity for the chemical technique for inorganic phosphate. The results do nevertheless indicate a maximum value for the concentration of inorganic PP, in parotid saliva. TABLE~.PP, ANDP,
INNORMALHIJMANSALIVAAND
DENTALCALCULUS
Whole Saliva
ws PP per mg PI
8 N-M
PI’ (PM) Mean
Range
Mean
Range
0.92
0.63-1.20
4.7
4.2-5.2
0*0!3&49
3.8
3-O-4.6
Parotid saliva
$601
0.26
calculus
(20%)
-
-
-
-
Mean
Range
4.4
2.5-6.3
The concentration of PPi measured in stimulated parotid saliva is about one thousand times less than that obtained by VOGEL and AMDUR (1967). This difference is in all probability due to a difference in the specificity of the pyrophosphate method. Their method relies on colour differences at two different times after addition of acid molybdate, whereas ours includes steps to separate contaminating substances. Our results are more in agreement with those of JENKINS, FERGUSON and EDGAR (1967) and JENKINS and EDGAR (1970). It is unlikely that the different results are due to differences in flow rate. DAWES (1969) showed that the orthophosphate concentration was markedly dependent on flow rate and the orthophosphate concentrations obtained by us and by VOGEL and AMDUR (1967) are very similar. Our results suggest a higher concentration of PP, in whole saliva than in parotid saliva. Whether this difference is due to different flow rate conditions used or to a higher concentration of PP, in submaxillary saliva or to a contribution by oral bacteria cannot be answered by our data to date. It is interesting that the concentration of PP, in resting saliva is high enough for it to have a role in the regulation of mineralization of dental plaque (FLEISCH and RUSSELL, 1970). Our results give no clue concerning the origin of PPi in calculus. It may be derived from saliva, gingival crevice fluid, blood or bacterial metabolism within plaque or from combinations of any one of these potential sources.
1392
E. HAUSMANN, S. BI~AZ,R. G. G. RUWZLL AND H. FLEISCH
Acknowledgements-E.H. is grateful to Professor HERBERTFLEISCHfor the opportunity to work in his Institute and to Procter & Gamble Co. for a travel grant. This work was supported by United States Public Health Service Grants, DE-01932-06 and AM-07266, by Grant 4564 from the Schweizerischer National fonds zur FBrderung der wissenschaftlichen Forschung. We are grateful to Miss P. BRONZ for skilled technical help. REFERENCES BISAZ, S., RUSSELL,R. G. G. and FLEISCH,H. 1968. Isolation of inorganic pyrophosphate from bovine and human teeth. Archs oral Biol. 13,683-696. CHEN, P. S., TORIBARA,T. Y. and WARNER, H. 1956. Microdetermination of phosphorus. Anal. Chem. 28, 1756-1758. DAWES, C. 1969. The effect of flow rate and duration of stimulation on the concentrations of protein and the main electrolytes in human parotid saliva. Archs oral Biol. 14,277-294. FLEISCH,H. and NEUMAN,W. F. 1961. Mechanisms of calicification: role of collagen poiyphosphates, and phosphatase. Am. J. Physiol. 200, 1296-1300. FLEISCH, H. and RUSSELL,R. G. G. 1970. Pyrophosphate and polyphosphate. In: The fnternational Encyclopedia of Pharmacology and Therapeutics, Section 51 (edited by PETERS,G. and RADOUCOTHOMAS,C.) Pergamon Press, Oxford. Chap. 3. In press. FLEISCH, H., RUSSELL,R. G. G. -and STRAUMANN,F. 1966. Effect of pyrophosphate on hydroxyapatite and its implications in calcium homeostasis. Nature, Land. 212,901-903. JENKINS,G. N., FERGUSON,D. B. and EDGAR, W. M. 1967. Fluoride and the metabolism of salivary bacteria. Helv. odont. Acta 11, 2-10. JENKINS,G. N. and EDGAR, W. M., 1970. Pyrophosphate in saliva and plaque. Abstract No. 232, I.A.D.R. 48th meeting. KUNITZ, M. 1961. An improved method for isolation of crystalline pyrophosphatase from bakers yeast. Archs biochem. Biophys. 92,210-272. RUSSELL,R. G. G., Brs~z, S., DONATH,A. and FLEISCH,H. 1970. Inorganic pyrophosphate in plasma in normal persons and in patients with hypophosphatasia, osteogenesis imperfecta and other disorders of bone. J. clin. Invest. In press. UNEMOTO,Y. 1961. Germ-free human parotid saliva. J. Osaka odont. Sot. 14, 14-21. VOGEL, J. J. and AMDUR, B. H. 1967. Inorganic pyrophosphate in parotid saliva and its relation to calculus formation. Archs oral Biol. 12, 159-163.