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Inorganic pyrophosphate in parotid saliva and its relation to calculus formation
Arrhsor&Viol.Vol.12,pp.159-l 63,1967. Pergamon Press Ltd. Printed in Gt.
Britain.
INORGANIC PYROPHOSPHATE IN PAROTID SALIVA AND ITS RELATION TO CALCULUS FORMATION J. J. VOGEL* and B. H. AMDUR Forsyth Dental Center and Harvard School of Dental Medicine, Boston, Massachusetts, U.S.A. Summary-The presence of inorganic pyrophosphate has been demonstrated in human parotid duct saliva by calorimetric analysis and paper chromatography. Preliminary studies suggest that the concentration of inorganic pyrophosphate in an individual’s saliva is a variable related to calculus forming status. INTRODUCTION MOST APPROACHES to
the study of dental calculus have involved causative factors and little attention has been devoted to the investigation of possible naturally occurring inhibitory mechanisms. Recently, FLEISCH and BISAZ (1962) found a material in normal human urine which was effective as an inhibitor of calcification in vitro. This material was identified as inorganic pyrophosphate, and authentic inorganic pyrophosphate was shown to have the same inhibitory effect. The demonstrated caries reducing effect in rodents (NIZEL and HARRIS, 1960; KOENIG,MARTHALERand M~~HLEMANN, 1961) of inorganic pyrophosphate and other polyphosphates suggests that interaction between these compounds and hydroxyapatite might contribute to the observed results. Such an interaction would also explain the inhibitory effect found by FLESH and BISAZ (1962). AMDUR et al. (1963) studied the effects of inorganic pyrophosphate and hexametaphosphate on synthetic hydroxyapatite in an attempt to elucidate the mechanism of this interaction. Both compounds reduced the acid solubility of hydroxyapatite and also inhibited its homogeneous nucleating ability in a metastable calcifying solution. These effects were produced either when hydroxyapatite was pretreated or when the polyphosphates were added directly to the calcifying solution. Amdur has postulated an interaction between the polyphosphates and the less restrained columnar calcium ions on the apatite surface. The occurrence of inorganic pyrophosphate in a biological fluid (urine), and the demonstration of its inhibitory effect on calcification prompted us to investigate this compound in saliva in relation to calculus deposition. EXPERIMENTAL
PROCEDURE
Parotid saliva was collected from the following three goups. Group I,-twelve children under 12 years of age; Group II,-eleven adults over 20 years of age with no clinical calculus problem; and Group III,-fifteen adults over 20 years of age with *Present address: University of Minnesota,
Medical School, Minneapolis, 159
Minn, U.S.A.
160
J. J. VOGEL AND B. H. AMDUR
a calculus problem requiring frequent clinical treatment. The children were included in the study because supragingival calculus deposition rarely occurs in this age group. Duct saliva was selected for the investigation in order to minimize extraneous factors found in whole saliva, such as contributions from oral flora and mammalian cell activity. RAPP, PRAPUOLENIS and MADONIA(1960) have shown samples of human whole saliva to contain considerable amounts of pyrophosphatase activity. Samples were collected during lime Life-saver stimulation using a modified Carlson-Crittenden device (SHANNON,PRIGMOREand CHAUNCEY,1962). For phosphate analysis, 1 ml of saliva was mixed with 1 ml of bovine serum albumin solution (1 mg/ml) and permitted to stand for 5 min, at which time 1 ml of O-2 M silicotungstic acid in 05 N H,SO* was added. The solution was mixed thoroughly and centrifuged at 3000 rev/min for 3-5 min. The serum albumin was added to facilitate complete precipitation of the more soluble salivary muco-proteins. Aliquots of the supernatant were analysed for ortho- and pyrophosphate by the method of FLYNN,JONESand LIPMANN,(1954). The values for orthophosphate in stimulated parotid saliva were comparable to those obtained by the method of LOWRY and LOPEZ(1946), which is highly specific for inorganic orthophosphate. The presence of inorganic pyrophosphate was confirmed by paper chromatography. Prior to chromatography, the saliva was decalcified with Dowex 50 - x 8, Na+ cycle ion exchange resin, ultrafIltered through a collodion membrane and concentrated lo-fold. Approximately 20 ~1 of the concentrate was applied to Whatman No. 1 TABLE 1. INORGANICPHOSPHATES IN PAROTIDSALIVA
mv-1 Group ~~~~
Number
~
I
~~
12 11 15
II III
(Paa’-)
(PO,‘-)
(P&‘-1 (mM) 3.57 f 0.72” 3.82 f O-20 4.69 f 1.95
(mM) 0.39 + o-09 O-42 f 0.07 0.27&0*13
Ratio
9.3 f 1.33 9.3 + 1.65 20.3 19.05
*Standard deviation.
paper and the chromatogram developed ascending in isopropyl ether-go% formic acid (30:20). Phosphate compounds were detected using the method of BANDERSKI and AXELROD(1951). Both a simultaneously run standard reference spot and a recovery standard of saliva enriched with authentic pyrophosphate were used to co&m the presence of inorganic pyrophosphate in the original saliva sample. The phosphate ester O-phosphorylethanolamine was also detected using this method of chromatography. This compound and a mixture of a-and-fl-glycerophosphates did not interfere in the analytical method of FLYNNet al. (1954). RESULTS AND DISCUSSION orthophosphate could compete with pyrophosphate for mineral sites to alter the effects of the later on calculus deposition, the ratio of [Pi] to [P N Pi] is Since
INORGANIC
PYROPHOSPHATE
IN PAROTlD
SALIVA
AND ITSRELATIONTO CALCULUS FORMATION 161
included. The variation in individual flow rates during sample collection was minimized by the approximately maximal flow afforded by the stimulus employed. Little or no difference was found between the data on saliva from the children and the non-calculous adults. The average concentration of pyrophosphate was O-4 mM. This level is one-tenth of the amount found by RMP et al. (1960) to produce dental erosion. All three values determined were different in the saliva from known calculus formers. The orthophosphate concentrations were higher and the pyrophosphate concentrations were lower than in the saliva samples from both children and non-calculous adults. The higher orthophosphate and lower pyrophosphate concentrations resulted in a higher PO,:P,O, ratio for this group. Statistical analysis of the data (Table 2) showed that higher orthophosphate concentration in the calculus TABLET. ANALYSISOFVARUNCEAMONG
GROUPS
p value* Orthophosphate Pyrophosphate
PO,: P40, ratio
IandII I and III II and IIl IandII IandIII II and III IandII IandIII II and JII
< o-05 < 0.01 < 0.05 o-40 < o-02 < 0.01 > 1.0 < om1 < 0401
*Calculated using short-range Student “t” test,
forming adults was significantly different from the concentration in children’s saliva, but not from that of the non-calculous adults. The levels of pyrophosphate in saliva from calculus forming adults were significantly lower than the levels in saliva from either the children or the non-calculous adults. The differences in the POI:P,O, ratios were highly significant on a similar comparison. RAPP et al. (1960) failed to find any inorganic pyrophosphate in whole human saliva, unless pyrophosphatase inhibitors, such as F- or hydroxylamine, were added, and stated that its accumulation was dependent upon microbial synthesis. The data on the phosphate partioning in saliva published by LURA (1947) do not exclude inorganic pyrophosphate, which would appear in any of his acid hydrolysed fractions as orthophosphate, and hence, be indistinguishable from organic phosphate esters. In the present work, duct saliva rather than whole saliva was subjected to analysis for inorganic pyrophosphate by a method relatively specific for it. The quantitative analysis was corroborated by the qualitative demonstration of the presence of inorganic pyrophosphate in our material by paper chromatography. Our results confirm the findings of SAWINSKI and COLE (1965) on the presence of inorganic pyrophosphate in human parotid duct saliva. To the contrary, we arrive at slightly different correlations between parotid duct saliva pyrophosphate content and clinical calculus status. This difference could derive in part from our inclusion of M
J. J.
162
VOGEL
AND
B. H.
iiMDUR
younger age group which may represent a condition similar to non-calculous adults. Another source of difference may be attributable to our choice of analytical methods which would conceivably be influenced by salivary phosphate compounds other than the major ones tested for interference. The results of this study suggest a relationship between the levels of pyrophosphate in salivary secretions and the tendency to deposit calculus. The presence of inorganic pyrophosphate in duct saliva is not surprising since it is an end product of several metabolic pathways that may exist in salivary glands. For example, in protein synthesis, one molecule of inorganic pyrophosphate is released for each amino acid activated by adenosine triphosphate for incorporation into protein. Of interest also in the case of salivary secretions are the synthesis of mucopolysaccharides, since one molecule of inorganic pyrophosphate is released for each molecule of sugar phosphate activated for polymerization by uridine triphosphate, and the pyrophosphate yielding activation of sialic acid by cytidine triphosphate. In view of the cell membrane activities of the salivary gland in ion transport involving phospholipids, the cytidine triphosphate activation of 0-phosphorylethanolamine to yield cytidine diphosphoethanolamine and pyrophosphate may also be noted. Whether the differences found here reflect an increase in glandular inorganic pyrophosphatase activity in the calculus former; a change in membrane transport or resorption ability; or a change in metabolic level, may not yet be stated. In light of the recent report of FLEISCH,BISAZand CARE(1964) on increased urinary inorganic pyrophosphate resulting from dietary orthophosphate supplements, dietary status of an individual as a determinant in our observation, may not be excluded. Further work is required to clarify mechanism as well as to confirm these results. a
Acknowledgement-We acknowledge with gratitude the co-operation of Dr. J. who was able to so motivate his patients that they presented themselves as volunteer saliva donors. This investigation was supported in part by U.S. Public Health Service Research Grant D-2183, from the National Institute of Dental Research, National Institute of Health, Bethesda, Maryland. BLOOM,
ResumLLa presence de pyrophosphate inorganique salive humaine, situee dans le canal de St&on, par chromatographie sur papier. Des etudes preliminaires en pyrophosphate inorganique dans une salive don&e la formation de tartre.
est mise en evidence darts la analyse colorimetrique et par indiquent que la concentration est variable et en rapport avec
Zusammenfassung-Mit Hilfe kolorimetrischer Analyse und Papierchromatographie ist die Anwesenheit anorganischen Pyrophosphats im Speichel des menschlichen ParotisAusftihrungsganges nachgewiesen worden. Vorlaufige Untersuchungen deuten darauf hin, dass die Konzentration anorganischen Phosphats im Speichel einer Person eine Variable darstellt, die mit der Zahnsteinbildung zusammenhlgnt.
INORGANICPYROPHOSPHATE IN PAROTIDSALIVAAND ITS RELATION TO CALCULUS
FORMATION
163
REFERENCES AMDUR, B. H., SPINELLI, M. A., MESSER, A. C. and BRUDEVOLD,F. 1963. Some interactions of hydroxylapatite with polymerized phosphate compounds. International Association for Dental Research, 41st General Meeting, Pittsburgh, Abstract 331. BANDURSKI,R. S. and AXELROD, B. 1951. The chromatographic identification of some biologically important phosphate esters. J. biol. Chem. 193,405-410. FLEISCH, H. and BISAZ, S. 1962. Isolation from urine of pyrophosphate, a calcification inhibitor.
Am. J. Physiol. 203. 671-675. FLEIXH, H., BISAZ, S. and CARE, A. D. Effect of orthophosphate on urinary pyrophosphate excretion and the prevention of urolithiasis. Lancet 16 May, 1065-1067. FLYNN, R., JONES, M. E. and LIPMANN, F. 1954. A calorimetric determination of inorganic pyrophosphate. J. biol. Chem. 211, 791-796. KBNIG,K. G., MARTHALER, T. M. and M~~HLEMANN, H. R. 1961. Effects of some phosphates in a short-period rat caries experiment. Archs oral. Biof. 3, 258-270. LOWRY,0. H. and LOPEZ,J. A. 1946. The determination of inorganic phosphate in the presence of labile phosphate esters. J. biol. Chem. 162,421428. LURA, H. EGGERS 1947. Investigation on the salivary phosphates and phosphatases. J. dent. Res.
26,203-224. NIZEL, A. E., HARRIS, R. S. and PARKER, J. 1960. Effects of metaphosphoric acid supplementation on morphology and caries incidence of hamster molars. International Association for Dental Research, 38th General Meeting, Chicago, Abstract 206. NIZEL, A. E. and HARRIS, R. S. 1960. Phosphates and dental caries: 1. Effect of metaphosphoric acid in the diet of weanling hamsters on dental caries development. J. Amer. dent. Ass. 60,
193-196. RAPP, G. W., PRAPUOLENIS,A. and MADONIA, J. 1960. Pyrophosphate, a factor in tooth erosion. J. dent. Res. 39, 372-376. SAWMSKI, V. J. and COLE, D. F. 1965. Phosphate concentrations of sterile human parotid saliva and its relationship to dental disorders. J. dent. Res. 44, 827. SHANNON,I. L., PRICIMORE,J. R. and CHAUNCEY, H. H. 1962. Modified Carlson-Crittenden device for the collection of parotid fluid. J. dent. Res. 41, 778-783.
Britain.
INORGANIC PYROPHOSPHATE IN PAROTID SALIVA AND ITS RELATION TO CALCULUS FORMATION J. J. VOGEL* and B. H. AMDUR Forsyth Dental Center and Harvard School of Dental Medicine, Boston, Massachusetts, U.S.A. Summary-The presence of inorganic pyrophosphate has been demonstrated in human parotid duct saliva by calorimetric analysis and paper chromatography. Preliminary studies suggest that the concentration of inorganic pyrophosphate in an individual’s saliva is a variable related to calculus forming status. INTRODUCTION MOST APPROACHES to
the study of dental calculus have involved causative factors and little attention has been devoted to the investigation of possible naturally occurring inhibitory mechanisms. Recently, FLEISCH and BISAZ (1962) found a material in normal human urine which was effective as an inhibitor of calcification in vitro. This material was identified as inorganic pyrophosphate, and authentic inorganic pyrophosphate was shown to have the same inhibitory effect. The demonstrated caries reducing effect in rodents (NIZEL and HARRIS, 1960; KOENIG,MARTHALERand M~~HLEMANN, 1961) of inorganic pyrophosphate and other polyphosphates suggests that interaction between these compounds and hydroxyapatite might contribute to the observed results. Such an interaction would also explain the inhibitory effect found by FLESH and BISAZ (1962). AMDUR et al. (1963) studied the effects of inorganic pyrophosphate and hexametaphosphate on synthetic hydroxyapatite in an attempt to elucidate the mechanism of this interaction. Both compounds reduced the acid solubility of hydroxyapatite and also inhibited its homogeneous nucleating ability in a metastable calcifying solution. These effects were produced either when hydroxyapatite was pretreated or when the polyphosphates were added directly to the calcifying solution. Amdur has postulated an interaction between the polyphosphates and the less restrained columnar calcium ions on the apatite surface. The occurrence of inorganic pyrophosphate in a biological fluid (urine), and the demonstration of its inhibitory effect on calcification prompted us to investigate this compound in saliva in relation to calculus deposition. EXPERIMENTAL
PROCEDURE
Parotid saliva was collected from the following three goups. Group I,-twelve children under 12 years of age; Group II,-eleven adults over 20 years of age with no clinical calculus problem; and Group III,-fifteen adults over 20 years of age with *Present address: University of Minnesota,
Medical School, Minneapolis, 159
Minn, U.S.A.
160
J. J. VOGEL AND B. H. AMDUR
a calculus problem requiring frequent clinical treatment. The children were included in the study because supragingival calculus deposition rarely occurs in this age group. Duct saliva was selected for the investigation in order to minimize extraneous factors found in whole saliva, such as contributions from oral flora and mammalian cell activity. RAPP, PRAPUOLENIS and MADONIA(1960) have shown samples of human whole saliva to contain considerable amounts of pyrophosphatase activity. Samples were collected during lime Life-saver stimulation using a modified Carlson-Crittenden device (SHANNON,PRIGMOREand CHAUNCEY,1962). For phosphate analysis, 1 ml of saliva was mixed with 1 ml of bovine serum albumin solution (1 mg/ml) and permitted to stand for 5 min, at which time 1 ml of O-2 M silicotungstic acid in 05 N H,SO* was added. The solution was mixed thoroughly and centrifuged at 3000 rev/min for 3-5 min. The serum albumin was added to facilitate complete precipitation of the more soluble salivary muco-proteins. Aliquots of the supernatant were analysed for ortho- and pyrophosphate by the method of FLYNN,JONESand LIPMANN,(1954). The values for orthophosphate in stimulated parotid saliva were comparable to those obtained by the method of LOWRY and LOPEZ(1946), which is highly specific for inorganic orthophosphate. The presence of inorganic pyrophosphate was confirmed by paper chromatography. Prior to chromatography, the saliva was decalcified with Dowex 50 - x 8, Na+ cycle ion exchange resin, ultrafIltered through a collodion membrane and concentrated lo-fold. Approximately 20 ~1 of the concentrate was applied to Whatman No. 1 TABLE 1. INORGANICPHOSPHATES IN PAROTIDSALIVA
mv-1 Group ~~~~
Number
~
I
~~
12 11 15
II III
(Paa’-)
(PO,‘-)
(P&‘-1 (mM) 3.57 f 0.72” 3.82 f O-20 4.69 f 1.95
(mM) 0.39 + o-09 O-42 f 0.07 0.27&0*13
Ratio
9.3 f 1.33 9.3 + 1.65 20.3 19.05
*Standard deviation.
paper and the chromatogram developed ascending in isopropyl ether-go% formic acid (30:20). Phosphate compounds were detected using the method of BANDERSKI and AXELROD(1951). Both a simultaneously run standard reference spot and a recovery standard of saliva enriched with authentic pyrophosphate were used to co&m the presence of inorganic pyrophosphate in the original saliva sample. The phosphate ester O-phosphorylethanolamine was also detected using this method of chromatography. This compound and a mixture of a-and-fl-glycerophosphates did not interfere in the analytical method of FLYNNet al. (1954). RESULTS AND DISCUSSION orthophosphate could compete with pyrophosphate for mineral sites to alter the effects of the later on calculus deposition, the ratio of [Pi] to [P N Pi] is Since
INORGANIC
PYROPHOSPHATE
IN PAROTlD
SALIVA
AND ITSRELATIONTO CALCULUS FORMATION 161
included. The variation in individual flow rates during sample collection was minimized by the approximately maximal flow afforded by the stimulus employed. Little or no difference was found between the data on saliva from the children and the non-calculous adults. The average concentration of pyrophosphate was O-4 mM. This level is one-tenth of the amount found by RMP et al. (1960) to produce dental erosion. All three values determined were different in the saliva from known calculus formers. The orthophosphate concentrations were higher and the pyrophosphate concentrations were lower than in the saliva samples from both children and non-calculous adults. The higher orthophosphate and lower pyrophosphate concentrations resulted in a higher PO,:P,O, ratio for this group. Statistical analysis of the data (Table 2) showed that higher orthophosphate concentration in the calculus TABLET. ANALYSISOFVARUNCEAMONG
GROUPS
p value* Orthophosphate Pyrophosphate
PO,: P40, ratio
IandII I and III II and IIl IandII IandIII II and III IandII IandIII II and JII
< o-05 < 0.01 < 0.05 o-40 < o-02 < 0.01 > 1.0 < om1 < 0401
*Calculated using short-range Student “t” test,
forming adults was significantly different from the concentration in children’s saliva, but not from that of the non-calculous adults. The levels of pyrophosphate in saliva from calculus forming adults were significantly lower than the levels in saliva from either the children or the non-calculous adults. The differences in the POI:P,O, ratios were highly significant on a similar comparison. RAPP et al. (1960) failed to find any inorganic pyrophosphate in whole human saliva, unless pyrophosphatase inhibitors, such as F- or hydroxylamine, were added, and stated that its accumulation was dependent upon microbial synthesis. The data on the phosphate partioning in saliva published by LURA (1947) do not exclude inorganic pyrophosphate, which would appear in any of his acid hydrolysed fractions as orthophosphate, and hence, be indistinguishable from organic phosphate esters. In the present work, duct saliva rather than whole saliva was subjected to analysis for inorganic pyrophosphate by a method relatively specific for it. The quantitative analysis was corroborated by the qualitative demonstration of the presence of inorganic pyrophosphate in our material by paper chromatography. Our results confirm the findings of SAWINSKI and COLE (1965) on the presence of inorganic pyrophosphate in human parotid duct saliva. To the contrary, we arrive at slightly different correlations between parotid duct saliva pyrophosphate content and clinical calculus status. This difference could derive in part from our inclusion of M
J. J.
162
VOGEL
AND
B. H.
iiMDUR
younger age group which may represent a condition similar to non-calculous adults. Another source of difference may be attributable to our choice of analytical methods which would conceivably be influenced by salivary phosphate compounds other than the major ones tested for interference. The results of this study suggest a relationship between the levels of pyrophosphate in salivary secretions and the tendency to deposit calculus. The presence of inorganic pyrophosphate in duct saliva is not surprising since it is an end product of several metabolic pathways that may exist in salivary glands. For example, in protein synthesis, one molecule of inorganic pyrophosphate is released for each amino acid activated by adenosine triphosphate for incorporation into protein. Of interest also in the case of salivary secretions are the synthesis of mucopolysaccharides, since one molecule of inorganic pyrophosphate is released for each molecule of sugar phosphate activated for polymerization by uridine triphosphate, and the pyrophosphate yielding activation of sialic acid by cytidine triphosphate. In view of the cell membrane activities of the salivary gland in ion transport involving phospholipids, the cytidine triphosphate activation of 0-phosphorylethanolamine to yield cytidine diphosphoethanolamine and pyrophosphate may also be noted. Whether the differences found here reflect an increase in glandular inorganic pyrophosphatase activity in the calculus former; a change in membrane transport or resorption ability; or a change in metabolic level, may not yet be stated. In light of the recent report of FLEISCH,BISAZand CARE(1964) on increased urinary inorganic pyrophosphate resulting from dietary orthophosphate supplements, dietary status of an individual as a determinant in our observation, may not be excluded. Further work is required to clarify mechanism as well as to confirm these results. a
Acknowledgement-We acknowledge with gratitude the co-operation of Dr. J. who was able to so motivate his patients that they presented themselves as volunteer saliva donors. This investigation was supported in part by U.S. Public Health Service Research Grant D-2183, from the National Institute of Dental Research, National Institute of Health, Bethesda, Maryland. BLOOM,
ResumLLa presence de pyrophosphate inorganique salive humaine, situee dans le canal de St&on, par chromatographie sur papier. Des etudes preliminaires en pyrophosphate inorganique dans une salive don&e la formation de tartre.
est mise en evidence darts la analyse colorimetrique et par indiquent que la concentration est variable et en rapport avec
Zusammenfassung-Mit Hilfe kolorimetrischer Analyse und Papierchromatographie ist die Anwesenheit anorganischen Pyrophosphats im Speichel des menschlichen ParotisAusftihrungsganges nachgewiesen worden. Vorlaufige Untersuchungen deuten darauf hin, dass die Konzentration anorganischen Phosphats im Speichel einer Person eine Variable darstellt, die mit der Zahnsteinbildung zusammenhlgnt.
INORGANICPYROPHOSPHATE IN PAROTIDSALIVAAND ITS RELATION TO CALCULUS
FORMATION
163
REFERENCES AMDUR, B. H., SPINELLI, M. A., MESSER, A. C. and BRUDEVOLD,F. 1963. Some interactions of hydroxylapatite with polymerized phosphate compounds. International Association for Dental Research, 41st General Meeting, Pittsburgh, Abstract 331. BANDURSKI,R. S. and AXELROD, B. 1951. The chromatographic identification of some biologically important phosphate esters. J. biol. Chem. 193,405-410. FLEISCH, H. and BISAZ, S. 1962. Isolation from urine of pyrophosphate, a calcification inhibitor.
Am. J. Physiol. 203. 671-675. FLEIXH, H., BISAZ, S. and CARE, A. D. Effect of orthophosphate on urinary pyrophosphate excretion and the prevention of urolithiasis. Lancet 16 May, 1065-1067. FLYNN, R., JONES, M. E. and LIPMANN, F. 1954. A calorimetric determination of inorganic pyrophosphate. J. biol. Chem. 211, 791-796. KBNIG,K. G., MARTHALER, T. M. and M~~HLEMANN, H. R. 1961. Effects of some phosphates in a short-period rat caries experiment. Archs oral. Biof. 3, 258-270. LOWRY,0. H. and LOPEZ,J. A. 1946. The determination of inorganic phosphate in the presence of labile phosphate esters. J. biol. Chem. 162,421428. LURA, H. EGGERS 1947. Investigation on the salivary phosphates and phosphatases. J. dent. Res.
26,203-224. NIZEL, A. E., HARRIS, R. S. and PARKER, J. 1960. Effects of metaphosphoric acid supplementation on morphology and caries incidence of hamster molars. International Association for Dental Research, 38th General Meeting, Chicago, Abstract 206. NIZEL, A. E. and HARRIS, R. S. 1960. Phosphates and dental caries: 1. Effect of metaphosphoric acid in the diet of weanling hamsters on dental caries development. J. Amer. dent. Ass. 60,
193-196. RAPP, G. W., PRAPUOLENIS,A. and MADONIA, J. 1960. Pyrophosphate, a factor in tooth erosion. J. dent. Res. 39, 372-376. SAWMSKI, V. J. and COLE, D. F. 1965. Phosphate concentrations of sterile human parotid saliva and its relationship to dental disorders. J. dent. Res. 44, 827. SHANNON,I. L., PRICIMORE,J. R. and CHAUNCEY, H. H. 1962. Modified Carlson-Crittenden device for the collection of parotid fluid. J. dent. Res. 41, 778-783.