Magnesium in human saliva

At&s oral Bid.

Vol. 13,pp. 1311-1319,1968.

Pcrgamon Pnu. Printedin

MAGNESIUM

at. Britain.

IN HUMAN SALIVA

R. D. LEAR* and P. GRIN Forsyth Dental Center, Boston, Massachusetts 02115, U.S.A. Summary-Magnesium and calcium concentrations were measured by atomic absorption spectrophotometry in resting and stimulated parotid and submandibular saliva. Magnesium concentration and flow rate were inversely related. Resting parotid saliva averaged O-131 mM Mg (flow rate 0.05 ml/min) and stimulated parotid saliva contained O-012 mM (flow rate 1.25 ml/min). Corresponding values for submandibular saliva were 0.098 and O-030 mM for flow rates of 0.07 and 1.11 ml/min respectively. The data correlate with the fmding that calculus from the lower anterior teeth contains significantly less Mg than maxillary molar deposits. Magnesium in parotid and submandibular saliva was fully ultraliltrable whereas Ca was 45-90 per cent ultraiiltrable. In whole saliva 61-70 per cent of the Mg was ultrafiltrable. It is suggested that the inclusion of cells in whole saliva accounts for the latter finding.

INTRODUCTION INORGANIC constituents of supragingival calculus are supplied by saliva (LEUNG, 1960) and such deposits in the maxillary molar region have a higher magnesium content than supragingival calculus found on mandibular anterior teeth (GRIN, VAN CAMPENand LINDSTROM,1967). It is hypothesized that the latter finding may be a reflection of localized differences in salivary magnesium concentration. Previous studies have generally considered only the magnesium content of whole saliva, the values reported showing wide variations. Thus, CLARKet al. (1927) found an average concentration of 0 -28mM and a range from 0 -08 to O-53 mM. BECKS(1928) reported whole saliva to contain 0 - 53 mM of magnesium. KRASNOW,OBLATTand FRIEDSON(1938) observed magnesium concentrations to be between 0 - 19 and 0.30 mM. ERICSSON(1955) stated that resting whole saliva contained 0 *23 mM and stimulated whole saliva 0 * 18 mM. Gow (1965a) examined 67 samples of resting saliva and 116 stimulated samples and found mean magnesium concentrations of O-206 and O-145 mM respectively. He also observed that secretion rate and magnesium concentration were inversely related. Reports on magnesium levels at the oral duct orifice are also at variance. ESCHLER, OCHSand SCHILLI(1965) reported resting parotid and submandibular saliva to contain 0 *55 and 0 -28mM respectively. Their corresponding values for stimulated duct fluid were 0 *33 and 0 - 20 mM. Recently WINDELERand SHANNON(1967) and DAWES(1967) studied the effect of flow rate on the magnesium concentration of parotid saliva. Low flow rate secretions were found to contain 0 - 175 and 0 - 155 mM respectively, and in highly stimulated saliva identical values of O-015 mM were reported. *Present Canada.

address:

Faculty of Dentistry,

University 1311

of British Columbia,

Vancouver

8, B.C..

1312

R. D. LEARAND P. GRBN

The present study was undertaken in view of the variation in values for whole saliva reported by different workers and because of the paucity of data on magnesium levels in the secretions from the two glands. The objective was to determine the nature of the relationship between magnesium concentration and flow rate in parotid and in submandibular secretions at their point of entry to the oral cavity (designated “duct saliva” in this paper). Additionally, calcium levels were studied for comparative purposes and some saliva samples were ultrafiltered to assess the extent of magnesium and calcium binding to large molecules. METHODS

AND

MATERIALS

Saliva for all the experiments was obtained from male and female volunteers whose ages ranged from 20 to 40 yrs. In every case the subjects were seated comfortably in a relatively quiet environment. Parotid samples were collected by a Carlson-Crittenden cup as modified by SHANNON, PRIGMOREand CHAUNCEY(1962). Wharton’s duct was cannulated with a polyethylene tube. For simultaneously collected saliva, individual samples of 3-4 ml were collected from the two glands of thirteen subjects, of whom seven were also available to participate in a subsequent examination of unstimulated (“resting”) secretions. High glandular outputs (“stimulated” saliva) were induced by having the subjects suck a lime Life Saver. Data from these experiments using two levels of salivary output suggested that it would be desirable to investigate concentrations at three or more flow rates. To this end, samples were collected (a) in the “resting” condition, (6) when the subject placed a lime Life Saver on his tongue, and (c) when vigorously chewing two lime Life Savers. Parotid data were obtained from 22 volunteers and again, seven were still available to give submandibular samples. Submandibular saliva was examined at an additional secretory level by medicating four subjects with O-2 mg atropine. Mean secretion rates under each of the foregoing conditions were calculated by collecting the sample in a graduated centrifuge tube over measured lengths of time. Calcium and magnesium concentrations were determined immediately after sample collection by atomic absorption spectrophotometry as discussed by Gow (1965 a, b). Lanthanum oxide (American Potash and Chemical Corp., Chicago, Ill.) which was essentially free of magnesium and calcium was used to prevent phosphate interference. Ultrafiltration required 12-15 ml samples of both duct and whole saliva. These were obtained from 12 subjects. The pH range was held at 6.8 to 7.2 by running a water-saturated mixture of 8.2 per cent CO2 in Nz over the surface. After removing an aliquot for spectrophotometric analyses, duplicate amounts of 4 ml were placed in sacs formed from 9 cm lengths of size 20 dialysis tubing (Union Carbide Corp., Chicago, Ill.) previously soaked in a warm 1 per cent potassium bicarbonate solution followed by extensive washing in distilled and deionized water. Before use the sac was rinsed with l-2 ml saliva. The sac was placed in a TORIBARA(1957) tube and further gassed for 5 min prior to centrifugation for 90 min at 2000 rev/min at room temperature. The yield of ultrafiltrate was 0 -5-O- 7 ml of which 0 -4 ml was used for analysis.

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MAONBsluMrNHuMANsALlvA

RESULTS

In the first series of experiments, magnesium values were recorded from simultaneously collected saliva at two levels of flow. Stimulated parotid saliva contained about l/3 less magnesium than the corresponding submandibular secretions, (O-023 vs. O-036 mM) whereas the magnesium content of unstimulated parotid saliva (0.13 1 mM) was 1.5 times greater than that from the submandibular gland (0.087 mM). However, it should be noted that in the “resting” state, parotid flow is only one third of the submandibular output, but when stimulated the two glands secrete at a similar rate (Table 1). Calcium concentration in unstimulated saliva from both glands was almost the same, but stimulated parotid saliva had only one half the calcium concentration of the corresponding submandibular secretion. It should be noted that the glandular outputs vary widely among subjects as indicated by the ranges given. When the influence of flow rate was further studied by using three levels of stimulation, it was again found that between-subject variations in output were substantial (Table 2). Nevertheless, when mean figures are considered, it is seen that progressive stimulation of both the glands still produced a diminution in the magnesium concentration. Calcium concentration was virtually unchanged when parotid flow changed from 0.24 to 1.25 ml/min. Submandibular calcium levels rose markedly when oral stimulation was increased. The very slow secretions produced by atropinized subjects were notably high in magnesium and calcium. The above trends are represented in Fig. 1. TABLE 1. MAGNESIUMAND CALCIUMCONCENIRATIONS IN SIMULTANEXJSLY COLIJKTED DUCT SALIVA

Stimulated Parotid

Mg (mM) Ca (mM) Flow ml/mint Range N

0.023 *0*012* 0.86 *0*033 0.50 0~11-0*75 13

Submandibular 0.036 *to*020 1.58 *O-286 0.54 0.23-1.30 13

Unstimulated Parotid Submandibular 0.131 *to*079 1.28 *0*544 0.05 0*01-0~10 7

0.087 io.061 1.58 zko.803 0.12 0*03-0*22 7

l Standard deviation. t For one gland.

The findings from the ultrafiltration experiments are summarized in Table 3. It is seen that both in parotid saliva (twelve samples in duplicate) and in submandibular saliva (eight samples in duplicate) the magnesium was 100 per cent ultrafihrable as opposed to calcium of which only 57-85 per cent (parotid) and 45-91 per cent (submandibular) passed through the membrane. Five samples of whole saliva were also examined. It may be noted that the mean magnesium content was higher in these samples and furthermore magnesium ultrafiltrability ranged from 60 to 70 per cent. A.0.A 10/l 1-B

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R. D. LEAR AND P. GRP~N TABLE2. MAGNE~KIMAND CALCIUMIN DUIZXSALIVAAT

DIFFERENT

FLOWRATES

PAROTID Stimulation

Flow rate

level

ml/mm*

Unstimulated (“resting”) Life Saver on tongue Chewing Life Saver

Magnesium concentrations mM

calcium concentration mM Mean S.D.

N

Mean

Range

Mean

S.D.

0.24

0.01-0.87

0.036

0.019

0.78

0.25

22

0.70 1.25

o-05-1.60 0.56-3.00

0.018 0.012

0.009 0.010

0.76 0.84

0.23 0.21

22 22

SUBMANDIBULAR Stimulation level Atropinized Unstimulated (“resting”) Life Saver on tongue Chewing Life Saver

Flow rate ml/min*

Magnesium concentrations

Calcium mM

concentration

mM

Mean

Range

Mean

S.D.

Mean

SD.

0.07 0.14 0.57 1.11

0~05-0*10 0*07-0*29 o-30-1.00 0.48-1.66

0.098 0.044 0.036 0.030

0.043 0.018 0.010 0.010

1.88 1.39 1.51 1.82

0.56 0.35 0.50 0.43

* For one gland.

FIG. 1. Mean concentrations

of calcium and magnesium in parotid and submandibular saliva at various flow rates.

N 4 7 7 7

l

Standard deviation.

saliva

WhOk

Submandibular saliva

S&liVt%

Parotid

-.

5

8

12

N

0.029 @013 0.043 40*027 0.087 &to*045

0.029

Mg in uhfikrate (mW

&o*Oi3* 0.043 ho.027 0.132 &0*068

Mg original (mW % of

(range6;8-108) (range 61-70)

Origilld

@38 1.76 jO.80 l-48 +0*80

0.92

@W

ca original

*o* 26 1.30 k.0~62 1.05 f0.55

o-70

Ca in ultrafiltrate (mM)

% of Origid

4

fi

g

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R. D. LEARANDP. GRBN

DISCUSSION The chemical findings presented here for calcium and magnesium levels in parotid saliva are in the range of those reported by WrNnELnRand SHANNON(~967)and by DAWE~ (1967). However, we did not find a marked increase in calcium with increasing flow as found by these authors. On the other hand, our data showing an inverse relationship between flow rate and magnesium concentration in both parotid and submandibular saliva are in accord with the observations of ERICSSON (1955) and of Gow (1965a) for whole saliva and of WINDELERand SHANNON(1967) and DAWE~(1967) for parotid saliva. However, the magnesium levels we have found differ considerably from those observed by EXHLER et al. (1965). Whether their high values may be attributed to the calorimetric method employed, the accuracy of which has been questioned (WACKER and VALLEE,1958), or whether they reflect geographical differences is not known. Recent evidence suggests that plasma magnesium may show substantial seasonal, climatic or regional differences (WALSEX,1967). It is possible that salivary magnesium may vary similarly. The different chemical composition of supragingival calculus from maxillary molars and mandibular incisors (GRBNet al., 1967) could be explained by the influence on their chemical microenvironment of secretions from the contiguous salivary ducts. It is suggested that the high magnesium concentration in resting parotid secretions results in the observed high magnesium content of calculus found near Stensen’s duct. Conversely, the lower values of magnesium found in mandibular anterior calculus may be due to the lesser magnesium content of secretions from the unstimulated submandibular gland. Findings of SCHNEYERet al. (1956), and LEAR, FLANAGANand Mao(1965) indicate that salivary flow is very low during sleep, and it is reasonable to assume that resting flow rates prevail for appreciable periods throughout the day. Thus, it is inferred that the chemical environment produced by such prolonged low salivary flow is a determining factor in calculus composition. Furthermore, the incorporation of magnesium into calculus may be facilitated by the level of calcium phosphate saturation which BRUDEVOLD, GRPINand MCCANN(1964) found to be low in resting parotid saliva. It should be noted that in the condition of “physiological rest”, submandibular flow is about three times greater than parotid. Yet, when submandibular output is pharmacologically decreased to approximately that of resting parotid flow, then the submandibular magnesium concentration approaches that of the resting parotid secretion. This indicates that there is a similar basic pattern of magnesium excretion in the two glands. However, even at low rates of flow, salivary magnesium concentration (0.13 mM) is much lower than that of serum (0.75 mM). In their study of parotid saliva, WINDELER and SHANNON(1967) pointed out that, although the concentration of salivary magnesium decreased with increase in flow, the output of magnesium per minute increased, and they suggested that simple diffusion could produce such a pattern. In the present study, parotid magnesium output varied from 0.012 wq. per minute for the lowest flow rate to 0.030 peq. for the highest flow rate. The corresponding values

MAGNESNM

IN HUMAN

SALIVA

1317

for submandibular saliva were O-014 and O-066 wq. Our findings are therefore in accord with those of WINDELEXand SHANNON for parotid saliva, and further show that a similar condition applies to submandibular gland excretion. The higher concentration at low flow rates could be caused by re-absorption of water in the duct system. The finding that magnesium in stimulated duct saliva is 100 per cent ultrafiltrable in contrast to calcium, which is only partially ultrafiltrable, shows that the two ions are not equally bound to large organic molecules. The present results do not exclude the possibility that magnesium and calcium may be bound to proteins of low molecular weight, which could pass the ultrafiltration membrane. The fact that magnesium in whole saliva is not fully ultrafiltrable has been observed previously by GOW (1965c). Inspection of Table 3 will show that the magnesium content of whole saliva was in the range of O-13 mM, which is higher than in stimulated duct saliva. The flow rate for whole saliva was 1.0-l -8 ml per minute. Considering the 4 major glands secreting at the same rate (O-2-0-4 ml/gland/mm.) mean concentrations of about O-03 mM could be expected. It thus appears that magnesium in whole saliva must be derived in part from another source. Cellular elements occur in whole saliva and they constitute a likely supply since they are known to contain 5-10 fold more magnesium than extracellular fluid (AMDUR,1967). To test this hypothesis a few samples of whole saliva were collected and divided in two parts. One part was “cleared” by centrifugation to remove the cellular elements, then both the treated and the untreated sample were ultrafiltered. The cleared samples showed lower magnesium content than the uncleared samples (about 70 per cent) and furthermore were almost fully ultrafiltrable (90-100 per cent). In other experiments mixtures of submandibular and parotid duct saliva were ultrafiltered and showed the same pattern as did the separate secretions. Therefore it is concluded that non-ultrafiltrable magnesium in whole saliva is associated with cellular elements. The finding that magnesium in duct saliva is fully ultrafiltrable while calcium is not, is consistent with the observation that a positive correlation existed between parotid salivary protein and calcium content, and a negative correlation between protein and magnesium (WINDELERand SHANNON,1967). It further points to the differences between salivary and plasma proteins. Thus in plasma about 30 per cent of the magnesium is protein bound, only a slightly smaller proportion than that of calcium (WALSER,1967). Acknowledgements-This investigation was supported by Research Grant DE 2183 from the N.I.D.R., N.I.H., U.S. Public Health Education and Welfare. This study was undertaken while Dr. Rosemary Lear was a postdoctoral research fellow at the Forsyth Dental Center.

R&&-b

concentrations en magnesium et calcium sont determinkes par spectrophotometrie d’absorption atomique dans lea salives parotidienne et sowmaxillaire, obtenues au repos et apr& stimulation. La concentration en magnesium est inversement proportionnelle h la vitesse de secretion. La salive parotidienne au repos contient 0,131 mM Mg (vitesse de secretion 0,OS ml/min) alors que la salive parotidienne stimul&z contient 0,012 mM (vitesse de secretion 1,25 ml/mm). Les valeurs correspondantes

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R. D. LEARAND P. GRON pour la salive sous-maxillaire sont respectivement de 0,098 et 0,030 mM, pour des vitesses de s&r&ion de 0,07 et 1,11 ml/min. Ces r&+ultats concordent aver le fait que le tartre de dents incisives inf&ieures contient signiiicativement moins de Mg que le tartre des molaires sup&ieures. Le magnbium parotidien et sous-maxillaire est complhtement ultrafiltrable, alors que 45-90 pour cent de Ca seulement l’est. Dans la salive mixte, 61-70 pour cent du Mg est ultra6ltrable. Ce fait peut s’expliquer par la pr&nce de cellules dans la salive mixte. Zusammenfassung-Mit Hilfe der Atomabsorptions-Spektrophotometrie wurden die Magnesium- und Calciumkonzentrationen im Ruhespeichel und im stimulierten Speichel der Gl. parotis und der Gl. submandibularis bestimmt. Magnesiumkonzentration und FlieDrate verhielten sich zueinander umgekehrt proportional. Der ParotisRuhespeichel enthielt durchschnittlich 0,131 mM Magnesium (FlieDrate 0,05 ml/n@, stimulierter Parotisspeichel 0,012 mM (FlieDrate 1,25 ml/min). Die entsprechenden Werte fi.ir Submandibularspeichel betrugen 0,098 bzw. 0,030 mM Mg bei FlieDraten von 0,07 bzw. I,11 ml/min. Die Ergebnisse stimmen rnit der Beobachtung hberein, daD Zahnstein an unteren Frontzlihnen signifikant weniger Mg enthalt als Ablagerungen an den oberen Molaren. Das Magnesium im Parotis- und SubmandibularspeicheI envies sich als in vollem Umfange, Calcium jedoch nur zu 45-90 Prozent ulttiltrierbar. Im Vollspeichel waren 61-70 Prozent des Magnesiums ultrafiltrierbar. Fiir diesen letzteren Befund scheint die Zusammenbalhmg von Zellen verantwortlich zu sein.

REFERENCES AMDUR,B. H. 1967. In: Secrerory Mechanism of Sakary Gland& p. 113. (Edited by SCHNEYER, L. H. and SCHNEYER, C. A.) Academic, New York. BECKS.H. 1928. Electrolytes of saliva under normal and pathological conditions. Proc. Sot. em Biof. Med. X,93-95. _ BRUDEVOLD,F., GRIN, P. and MCCANN, H. G. 1964. Physico-chemical aspects of the enamel-saliva system. In: Advances in Fluoride Research and Dental Caries Prevention. DD. 63-78. Vol. 3. Proc. 1-lth Congress European Organization for Research on Fluorine and Dental Car&s Prevention, July, Sandefjord, Norway. CLARK,G. W., SHELL,J. S., JOSEPHSON,J. B. and STOCKLE,M. E. 1927. The influence of diet upon the inorganic constitutents of human saliva. D. Cosmos 69, X0-513. DAWIX~,C. 1967. The secretion of magnesium and calcium in human parotid saliva. Caries Res. 1, 333-342. ERICSSON,Y. 1955. Simplified methods for determination of calcium and magnesium in saliva. J. dent. Res. 34, 104-112. EKHLER, J., OCHS, G. and SCHKLI, W. 1965. Der Mg *+ Ionengehalt im Sekret der menschlichen Speicheldrusen. Archs oral Biol. 10,969-973. Gow, B. S. 1965a. Analysis of metals in saliva by atomic absorption spcctrophotometry II. Magnesium. J. dent. Res. 44, 890-894. Gow, B. S. 1965b. Analysis of metals in saliva by atomic absorption spectrophotometry I. Calcium. J. dent. Res. 44, 885-890. Gow, B. S. 1965~. Non-ultrfiltrable calcium and magnesium in human saliva. Archs oral Biof. 10, 15-22. GRIN, P., VAN CAMPEN,G. J. and LINDSTROM, I. 1967. Human dental calculus: inorganic chemical and crystallographic composition. Archs oral Hot. 12,829-831. KRA~NOW.F.. OBLA~. E. B. and FRIEDSON.S. 1938. Dental caries control within our reach. J. Am. dent. ks: 30, 1508-1526. LEAR,C. S. C., FLANAGAN,J. B. and MOORREES, C. F. A. 1965. The frequency of deglutition in man. Archs oral Biol. 10,83-99. LEUNG,S. W. 1960. Salivary calculus deposition. In: Calcification in Biological Systems. (Edited by SOGNNAES,R. F.) American Association for the Advancement of Science., Washington, D.C. pp. 307-322. SCHNEYER,L. H., PIGMAN,W., HANAHAN,L. and GILMORE,R. W. 1956. Rate of flow of human parotid, sublingual and submaxillary secretions during sleep. J. dent. Res. 35, 109-114.

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SHANNON, I. L. PRIGMORE, J. R. aad C~AUNCEY,H. H. 1962.Modified Carlson-Crittenden device for collection of parotid fluid. J. dent. Res. 41.778-783. TORIBARA,T., TEREPI& A. R. and DEWEY,P. A. 1957. The ultrafiltrable calcium of human serum. I. Ultrafiltration methods and normal values. J. clin. Invest. 36, 738-748. WACKER,W.E.C. and VALLEE,B. L. 1958. Magnesium metabolism. New Engl. J. Med. 259,432. WALSER,M. 1967. Magnesium Metabolism. p. 191-201 In: Reviews of Physiology, Biochemistry and Experimental Pharmacology. Vol. 59, Springer, New York.

WINDBLER, A. S. and SHANNON, I. L. 1967. Effect of flow rate on parotid fluid calcium, magnesium, and protein concentrations in man. Archs oral Biol. l&1063-1069.