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The flow-rate-dependent excretion of ionized calcium in pilocarpine-stimulated human submandibular saliva
Archs oral Bid. Vol. 28, No. IO, pp. 907-909, Printed in Great Britain. All rights reserved
0003-9969/83$03.00 + 0.00 Copyright (8 1983 Pergamon Press Ltd
1983
THE FLOW-RATE-DEPENDENT EXCRETION OF IONIZED CALCIUM IN PILOCARPINE-STIMULATED HUMAN SUBMANDIBULAR SALIVA H. Medizinische
MAIER*,
Universitatsklinik
C. TRIEBEL
Wiirzburg,
and A.
Josef Schneiderstr.
HEIDLAND
2, 87 Wiirzburg,
West Germany
Summary-In 10 healthy male subjects the total calcium concentration in submandibular saliva varied between 2.05 + 0.12 mmol/l and 2.48 k 0.1 mmol/min, and did not show a dependency on the flow rate. The salivary-ionized calcium increased significantly at flow rates between 0.1 and 1.3 mlimin (from 0.74 f 0.05 to 1.41 + 0.04 mmol/l) and even reached plasma levels.
INTRODUCTION
jects reclined in a dental chair. For collection of saliva, a specially-made polyethylene catheter was passed 15-20 mm into the Wharton duct and was connected via PVC tubing to a calibrated 2mlpipette. The filling time of the pipette was used to calculate the flow rate. Saliva flow was stimulated by a subcutaneous injection of pilocarpine (0.0 15mg/kg). The duration of pilocarpine action was about 40min. Within this time, 10-16 samples were collected and analysed immediately after the experiment. The measurement of ionized Ca was performed by the use of the optimized-flow multi-measuring system, armed with carrier-membrane disk-electrodes; these are characterized by streamlined elevations in the channel system, providing a flow low in stagnation and dead zones (Schindler et al., 1980). The calibration of the electrode has been described (Maier was et al., 1979). The total Ca concentration measured spectrophotometrically (Beckman’“). For the measurement of inorganic phosphate, the Harleco%-test was used. The measurement of total protein was performed according to Lowry et al. (1951).
There are several extensive studies of the dependency of concentrations of the main anions and cations on flow rate in human parotid and submandibular saliva (Dawes, 1969, 1974; Kreusser et al., 1972) but little information exists on the handling of ionized Ca, which may play a major role in the regulation of ductal transport systems in human salivary glands (Schneyer, 1974). The study of Vogel, Naujoks and Brudevold (1965) on the concentration of Ca ions in human parotid and submandibular saliva has for long provided the only data on this subject. However, that study, based on a small number of experiments, used a colorimetric method to measure Ca (Walser, 1961) which is less accurate than modern electrochemical methods. In 1979 our group studied the flow-rate-dependent excretion of ionized Ca in pilocarpine-stimulated parotid saliva (Maier et cd., 1979) using an electrochemical multi-measuring system (EMS) and a calcium-selective carrier-membrane disk-electrode (Schindler, 1977). However, the measurement of ionized Ca in human submandibular saliva with this experimental set-up presented difficulties. Mucous deposition in the channels of the EMS made accurate measurement impossible. A newly-developed version of the EMS, the optimized-flow EMS (Schindler et al., 1980) has solved this problem. We have studied flow-ratedependent excretion of Ca ions in pilocarpinestimulated human submandibular saliva using this system. METHODS
RESULTS
AND MATERIALS
Submandibular saliva was collected from IO healthy male volunteers after an overnight fast. To avoid circadian variations of saliva composition (Dawes, 1975), all experiments were carried out between 8.0&10.00 h. During the experiments, the sub*Present address: HNO-Universitgtsklinik, VoOstr. 5-7. 6900 Heidelberg. West Germany. 907
The mean concentrations of electrolytes and total protein in human submandibular saliva are listed in Table 1. At flow rates between 0.1 and 1.3 ml/min, ionized Ca concentration rose significantly from 0.74 + 0.05 to 1.42 + 0.04 mmol/l (p < 0.001). At higher flow rates (1.3-2.0 ml/min), the Ca* + -concentration in submandibular saliva decreased slightly (1.33 + 0.05 mmol/l; p < 0.04). The total Ca concentration did not show significant flow-rate-dependent variations and remained between 2.43 + 0.10 and 2.05 + 0.12 mmol/I. The concentration of inorganic phosphate decreased at flow rates between 0.1-0.9 ml/min, from 3.14 f 0.19 to 1.76 +_0.08 mmol/l (p < 0.001). There was no change of protein concentration at different flow rates.
H.
908 Table
1. The concentrations Flow rate (ml/min)
of total calcium, ionized calcium, inorganic phosphate and stimulated final submandibular saliva at different flow rates Calutal (mmol/l)
0.14.4 0.4-0.6 0.60.9
(n = 9) (n = 14) (n = 38)
2.43 + 0.10 2.05 +0.12 2.29 + 0.1 I
0.9-1.3
(n = 46)
2.07 I_ 0.08
1.3-2.0
(n = 26)
MAIEK et al.
2. I8 + 0.07
0.74 * 0.05 0.92 k 0.08 I.13 + 0.04
1.42_+0.04 I .25 + 0.04
p p p p p p
< 0.01 < 0.001 < 0.04
3.14*0.19 2.28 f 0. I9 1.76 rf- 0.08 2.18 f0.15 2.05 * 0.08
The results are given as mean k SEM (n = number of samples). The first The second p value is related to the preceding flow-rate range.
DISCUSSION
Calcium in saliva consists of three fractions, protein-bound, complex-bound and ionized. The ionized Ca is the biologically active and therefore most important fraction. The reported concentrations for total Ca in stimulated human parotid saliva vary between 0.70 and 1.75 mmol/l (Vogel et al., 1965; Kreusser et al., 1972; Maier et al., 1979) and those for ionized Ca between 0.35 and 0.90 mmol/l (Vogel et al., 1965; Maier et al., 1979). The excretion patterns of total Ca and of ionized Ca in parotid saliva are dependent on the flow rate of saliva (Kreusser et al., 1972; Maier et al., 1979). The excretion curves are characterized by an exponential increase in concentration at flow rates from 0.1-0.6 ml/min, followed by a plateau at higher flow rates (Maier et al., 1979). 44 to 54 per cent of the total calcium in human parotid saliva is ionized. The ratio between ionized and total Ca is not altered by the flow rate. Micropuncture experiments in the rat parotid gland provide a means of studying of Ca turnover in this gland. Mangos (1980) found plasma-like Ca concentrations in the primary fluid. During passage through the glandular duct system, salivary Ca concentration decreased significantly. Thus he concluded that the primary fluid Ca concentration is modified by reabsorption mechanisms located in the ductal system. Similar investigations of the submandibular gland have not to our knowledge been performed. The reported concentrations of total Ca in stimulated human submandibular saliva vary between 1.66 and 2.32 mmol/l and those of ionized Ca between 1.04 and 1.72 mmol/l (Vogel et al., 1965; Dawes, 1974). Dawes (1974) studied the excretion of total Ca in stimulated human submandibular saliva at flow rates of 1.O, 2.0 and 3.0 ml/min. The total Ca measured in these experiments varied between 1.66+ 0.42 and 2.13 f 0.26 mmol/l. Dawes found a positive correlation between flow rate and salivary calcium concentration, in contrast to the findings of Blomfield, Warton and Brown (1973), who were unable to demonstrate a flow-rate-dependent total Ca excretion in human submandibular saliva. The ionized Ca concentration in gustatorystimulated human submandibular saliva studied by Vogel et al. (1965) varied between 1.04 and 1.72 mmol/l. The small number of samples (six), however, do not allow any conclusions to be drawn
protein
Inorganic phosphate (mmol/l)
Calanlzed (mmol/l) ns ns ns ns ns ns ns
total
in pilocarpine-
Total protein (mg:‘;,)
p < 0.007 p < 0.001 p < 0.03 p < 0.003 p < 0.05 p c 0.002
108.29 + 9.03 I IS.63 + 9.81 134.71 + Il.60 128.7+ 131.20+
11.63 12.1
ns
p value is related to the lowest flow-rate
ns ns :: ns ns ns range.
concerning the effect of flow rate on salivary ionized Ca. The total Ca concentrations in pilocarpinestimulated human submandibular saliva described here agree with those reported by Vogel et al. (1965) and are slightly higher than those reported by Dawes (1974). In contrast to the studies of Dawes but in agreement with those of Blomfield et al. (1973) and Ferguson and Botchway (1979), the total-salivary Ca concentration was not affected by changes in flow rate in our experiments. The ionized Ca fraction in pilocarpine-stimulated submandibular saliva on the other hand showed a significant increase at flow rates between 0.1 and 1.3 ml/min and, in contrast to Ca* +-concentration in parotid saliva, reached plasma-like concentrations. These experiments do not allow us to draw definite conclusions about the regulation of Ca turnover in the human submandibular gland. For this, micropuncture and microperfusion experiments would be necessary. However, our results indicate that the submandibular gland seems able to keep the total Ca concentration in pilocarpine-stimulated final saliva almost constant at flow rates between 0.1 and 2.0 ml/min. The flow-rate-dependency of the ionized Ca might be produced by Ca-complexing mechanisms modifying fractional Ca distribution in final saliva. Inorganic phosphate, which decreased significantly as the flow rate increased, might play a role as such a complexing agent. Alterations of the proteinbound Ca fraction seem to be unlikely: significant flow-rate-dependent variations of protein concentration in submandibular saliva could not be proved in this investigation. An effect of the pH on the Ca binding capacity of salivary proteins was excluded by Vogel et al. (1965). Acknowledgetnent--Supported schungsgemeinschaft
by the Deutsche For-
SFB 92 Biomund.
REFERENCES
Blomfield J., Warton K. L. and Brown J. M. (1973) Flow rate and inorganic components of submandibular saliva in cystic fibrosis. Archs DD. Child. 48, 267-274. Dawes C. (1969) The effects of flow-rate and duration of stimulation on the concentration of protein and the main electrolytes in human parotid saliva. Archs oral Biol. 14, 217-294.
Dawes C. (1974) The effects of flow-rate and duration of stimulation on the concentration of protein and the main
Ionized Ca in human submandibular
saliva
909
saliva. Archs ornl Biol. 19, 887-985. Dawes C. (1975) Circadian rhythms in the flow-rate and
Schneyer L. H. (1974) Effects of calcium on Na/K transport by perfused main duct of rat submaxillary gland. Am. J.
composition of unstimulated and stimulated human submandibular saliva. J. Physiol. 244, 535-548. Ferguson D. B. and Botchway C. A. (1979) Circadian variations in flow rate and composition of human stimulated submandibular saliva. Archs oral Biol. 24, 4331137. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. G. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-215. Kreusser W.. Heidland A., Hennemann H., Wigand M. E. and Knauf H. (1972) Mono- and divalent electrolyte patterns, pCOZ and pH in relation to flow-rate in normal human parotid saliva. Eur. J. din. Invest. 2, 398406. Maier H.. Coroneo M. T., Antonczyk G. and Heidland A. (1979) The flow-rate dependent excretion of ionized calcium in human parotid saliva. Archs oral Biol. 24,
Schindler J. G. (1977) MultimeDsystem fur die elektrochemische Analyse stromender Flussigkeiten und Gase. Biomed. Techn. 22, 235-244. Schindler J. G., v. Giilich M., Maier H.. Stork G.. Schal W., Braun H.-E., Schmid W. and Karaschinski K.-D. (1980) Striimungsoptimiertes Multimeljsystem mit ionenselektiven Disk-Elektroden. Fresenius Z. rmal. (‘hem. 301, 41&413. Vogel J. J., Naujoks R. and Brudevold F. (1965) The effective concentrations of calcium and inorganic orthophosphate in salivary secretions. Archs ord Bid. 10, 523-534. Walser M. (1961) Ion association IV. Interactions between calcium. magnesium, inorganic phosphate. citrate and protein in normal human plasma. J. c/in. Irne.c/. 40, 723-730.
electrolytes in human submandibular
225-127.
Physiol. 226, 821-826.
0003-9969/83$03.00 + 0.00 Copyright (8 1983 Pergamon Press Ltd
1983
THE FLOW-RATE-DEPENDENT EXCRETION OF IONIZED CALCIUM IN PILOCARPINE-STIMULATED HUMAN SUBMANDIBULAR SALIVA H. Medizinische
MAIER*,
Universitatsklinik
C. TRIEBEL
Wiirzburg,
and A.
Josef Schneiderstr.
HEIDLAND
2, 87 Wiirzburg,
West Germany
Summary-In 10 healthy male subjects the total calcium concentration in submandibular saliva varied between 2.05 + 0.12 mmol/l and 2.48 k 0.1 mmol/min, and did not show a dependency on the flow rate. The salivary-ionized calcium increased significantly at flow rates between 0.1 and 1.3 mlimin (from 0.74 f 0.05 to 1.41 + 0.04 mmol/l) and even reached plasma levels.
INTRODUCTION
jects reclined in a dental chair. For collection of saliva, a specially-made polyethylene catheter was passed 15-20 mm into the Wharton duct and was connected via PVC tubing to a calibrated 2mlpipette. The filling time of the pipette was used to calculate the flow rate. Saliva flow was stimulated by a subcutaneous injection of pilocarpine (0.0 15mg/kg). The duration of pilocarpine action was about 40min. Within this time, 10-16 samples were collected and analysed immediately after the experiment. The measurement of ionized Ca was performed by the use of the optimized-flow multi-measuring system, armed with carrier-membrane disk-electrodes; these are characterized by streamlined elevations in the channel system, providing a flow low in stagnation and dead zones (Schindler et al., 1980). The calibration of the electrode has been described (Maier was et al., 1979). The total Ca concentration measured spectrophotometrically (Beckman’“). For the measurement of inorganic phosphate, the Harleco%-test was used. The measurement of total protein was performed according to Lowry et al. (1951).
There are several extensive studies of the dependency of concentrations of the main anions and cations on flow rate in human parotid and submandibular saliva (Dawes, 1969, 1974; Kreusser et al., 1972) but little information exists on the handling of ionized Ca, which may play a major role in the regulation of ductal transport systems in human salivary glands (Schneyer, 1974). The study of Vogel, Naujoks and Brudevold (1965) on the concentration of Ca ions in human parotid and submandibular saliva has for long provided the only data on this subject. However, that study, based on a small number of experiments, used a colorimetric method to measure Ca (Walser, 1961) which is less accurate than modern electrochemical methods. In 1979 our group studied the flow-rate-dependent excretion of ionized Ca in pilocarpine-stimulated parotid saliva (Maier et cd., 1979) using an electrochemical multi-measuring system (EMS) and a calcium-selective carrier-membrane disk-electrode (Schindler, 1977). However, the measurement of ionized Ca in human submandibular saliva with this experimental set-up presented difficulties. Mucous deposition in the channels of the EMS made accurate measurement impossible. A newly-developed version of the EMS, the optimized-flow EMS (Schindler et al., 1980) has solved this problem. We have studied flow-ratedependent excretion of Ca ions in pilocarpinestimulated human submandibular saliva using this system. METHODS
RESULTS
AND MATERIALS
Submandibular saliva was collected from IO healthy male volunteers after an overnight fast. To avoid circadian variations of saliva composition (Dawes, 1975), all experiments were carried out between 8.0&10.00 h. During the experiments, the sub*Present address: HNO-Universitgtsklinik, VoOstr. 5-7. 6900 Heidelberg. West Germany. 907
The mean concentrations of electrolytes and total protein in human submandibular saliva are listed in Table 1. At flow rates between 0.1 and 1.3 ml/min, ionized Ca concentration rose significantly from 0.74 + 0.05 to 1.42 + 0.04 mmol/l (p < 0.001). At higher flow rates (1.3-2.0 ml/min), the Ca* + -concentration in submandibular saliva decreased slightly (1.33 + 0.05 mmol/l; p < 0.04). The total Ca concentration did not show significant flow-rate-dependent variations and remained between 2.43 + 0.10 and 2.05 + 0.12 mmol/I. The concentration of inorganic phosphate decreased at flow rates between 0.1-0.9 ml/min, from 3.14 f 0.19 to 1.76 +_0.08 mmol/l (p < 0.001). There was no change of protein concentration at different flow rates.
H.
908 Table
1. The concentrations Flow rate (ml/min)
of total calcium, ionized calcium, inorganic phosphate and stimulated final submandibular saliva at different flow rates Calutal (mmol/l)
0.14.4 0.4-0.6 0.60.9
(n = 9) (n = 14) (n = 38)
2.43 + 0.10 2.05 +0.12 2.29 + 0.1 I
0.9-1.3
(n = 46)
2.07 I_ 0.08
1.3-2.0
(n = 26)
MAIEK et al.
2. I8 + 0.07
0.74 * 0.05 0.92 k 0.08 I.13 + 0.04
1.42_+0.04 I .25 + 0.04
p p p p p p
< 0.01 < 0.001 < 0.04
3.14*0.19 2.28 f 0. I9 1.76 rf- 0.08 2.18 f0.15 2.05 * 0.08
The results are given as mean k SEM (n = number of samples). The first The second p value is related to the preceding flow-rate range.
DISCUSSION
Calcium in saliva consists of three fractions, protein-bound, complex-bound and ionized. The ionized Ca is the biologically active and therefore most important fraction. The reported concentrations for total Ca in stimulated human parotid saliva vary between 0.70 and 1.75 mmol/l (Vogel et al., 1965; Kreusser et al., 1972; Maier et al., 1979) and those for ionized Ca between 0.35 and 0.90 mmol/l (Vogel et al., 1965; Maier et al., 1979). The excretion patterns of total Ca and of ionized Ca in parotid saliva are dependent on the flow rate of saliva (Kreusser et al., 1972; Maier et al., 1979). The excretion curves are characterized by an exponential increase in concentration at flow rates from 0.1-0.6 ml/min, followed by a plateau at higher flow rates (Maier et al., 1979). 44 to 54 per cent of the total calcium in human parotid saliva is ionized. The ratio between ionized and total Ca is not altered by the flow rate. Micropuncture experiments in the rat parotid gland provide a means of studying of Ca turnover in this gland. Mangos (1980) found plasma-like Ca concentrations in the primary fluid. During passage through the glandular duct system, salivary Ca concentration decreased significantly. Thus he concluded that the primary fluid Ca concentration is modified by reabsorption mechanisms located in the ductal system. Similar investigations of the submandibular gland have not to our knowledge been performed. The reported concentrations of total Ca in stimulated human submandibular saliva vary between 1.66 and 2.32 mmol/l and those of ionized Ca between 1.04 and 1.72 mmol/l (Vogel et al., 1965; Dawes, 1974). Dawes (1974) studied the excretion of total Ca in stimulated human submandibular saliva at flow rates of 1.O, 2.0 and 3.0 ml/min. The total Ca measured in these experiments varied between 1.66+ 0.42 and 2.13 f 0.26 mmol/l. Dawes found a positive correlation between flow rate and salivary calcium concentration, in contrast to the findings of Blomfield, Warton and Brown (1973), who were unable to demonstrate a flow-rate-dependent total Ca excretion in human submandibular saliva. The ionized Ca concentration in gustatorystimulated human submandibular saliva studied by Vogel et al. (1965) varied between 1.04 and 1.72 mmol/l. The small number of samples (six), however, do not allow any conclusions to be drawn
protein
Inorganic phosphate (mmol/l)
Calanlzed (mmol/l) ns ns ns ns ns ns ns
total
in pilocarpine-
Total protein (mg:‘;,)
p < 0.007 p < 0.001 p < 0.03 p < 0.003 p < 0.05 p c 0.002
108.29 + 9.03 I IS.63 + 9.81 134.71 + Il.60 128.7+ 131.20+
11.63 12.1
ns
p value is related to the lowest flow-rate
ns ns :: ns ns ns range.
concerning the effect of flow rate on salivary ionized Ca. The total Ca concentrations in pilocarpinestimulated human submandibular saliva described here agree with those reported by Vogel et al. (1965) and are slightly higher than those reported by Dawes (1974). In contrast to the studies of Dawes but in agreement with those of Blomfield et al. (1973) and Ferguson and Botchway (1979), the total-salivary Ca concentration was not affected by changes in flow rate in our experiments. The ionized Ca fraction in pilocarpine-stimulated submandibular saliva on the other hand showed a significant increase at flow rates between 0.1 and 1.3 ml/min and, in contrast to Ca* +-concentration in parotid saliva, reached plasma-like concentrations. These experiments do not allow us to draw definite conclusions about the regulation of Ca turnover in the human submandibular gland. For this, micropuncture and microperfusion experiments would be necessary. However, our results indicate that the submandibular gland seems able to keep the total Ca concentration in pilocarpine-stimulated final saliva almost constant at flow rates between 0.1 and 2.0 ml/min. The flow-rate-dependency of the ionized Ca might be produced by Ca-complexing mechanisms modifying fractional Ca distribution in final saliva. Inorganic phosphate, which decreased significantly as the flow rate increased, might play a role as such a complexing agent. Alterations of the proteinbound Ca fraction seem to be unlikely: significant flow-rate-dependent variations of protein concentration in submandibular saliva could not be proved in this investigation. An effect of the pH on the Ca binding capacity of salivary proteins was excluded by Vogel et al. (1965). Acknowledgetnent--Supported schungsgemeinschaft
by the Deutsche For-
SFB 92 Biomund.
REFERENCES
Blomfield J., Warton K. L. and Brown J. M. (1973) Flow rate and inorganic components of submandibular saliva in cystic fibrosis. Archs DD. Child. 48, 267-274. Dawes C. (1969) The effects of flow-rate and duration of stimulation on the concentration of protein and the main electrolytes in human parotid saliva. Archs oral Biol. 14, 217-294.
Dawes C. (1974) The effects of flow-rate and duration of stimulation on the concentration of protein and the main
Ionized Ca in human submandibular
saliva
909
saliva. Archs ornl Biol. 19, 887-985. Dawes C. (1975) Circadian rhythms in the flow-rate and
Schneyer L. H. (1974) Effects of calcium on Na/K transport by perfused main duct of rat submaxillary gland. Am. J.
composition of unstimulated and stimulated human submandibular saliva. J. Physiol. 244, 535-548. Ferguson D. B. and Botchway C. A. (1979) Circadian variations in flow rate and composition of human stimulated submandibular saliva. Archs oral Biol. 24, 4331137. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. G. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-215. Kreusser W.. Heidland A., Hennemann H., Wigand M. E. and Knauf H. (1972) Mono- and divalent electrolyte patterns, pCOZ and pH in relation to flow-rate in normal human parotid saliva. Eur. J. din. Invest. 2, 398406. Maier H.. Coroneo M. T., Antonczyk G. and Heidland A. (1979) The flow-rate dependent excretion of ionized calcium in human parotid saliva. Archs oral Biol. 24,
Schindler J. G. (1977) MultimeDsystem fur die elektrochemische Analyse stromender Flussigkeiten und Gase. Biomed. Techn. 22, 235-244. Schindler J. G., v. Giilich M., Maier H.. Stork G.. Schal W., Braun H.-E., Schmid W. and Karaschinski K.-D. (1980) Striimungsoptimiertes Multimeljsystem mit ionenselektiven Disk-Elektroden. Fresenius Z. rmal. (‘hem. 301, 41&413. Vogel J. J., Naujoks R. and Brudevold F. (1965) The effective concentrations of calcium and inorganic orthophosphate in salivary secretions. Archs ord Bid. 10, 523-534. Walser M. (1961) Ion association IV. Interactions between calcium. magnesium, inorganic phosphate. citrate and protein in normal human plasma. J. c/in. Irne.c/. 40, 723-730.
electrolytes in human submandibular
225-127.
Physiol. 226, 821-826.