Saturation of human saliva with calcium phosphates

Salivary saturation was calculated in respect to hydroxyapatite (HA), tricalcium phosphate (TCP), octocalcium phosphate (OCP) and dicalcium phosphate dihydrate (DCPD). The calculations were based on ionized calcium and ionized orthophosphate in resting parotid saliva and in stimulated parotid submaxillary and whole saliva. The findings snowed that samples of resting parotid saliva were least saturated and often only in respect to HA. Stimulated secretions were also saturated in regard to TCP and OCP. Some samples of stimulated submaxillary and whole saliva were also saturated in regard to DCPD, while only few samples were supersaturated in this regard.     

The flow-rate-dependent excretion of ionized calcium in human parotid saliva.

The total calcium content of parotid saliva is increased in various diseases. Because of methodological difficulties, little is known about the ionized calcium fraction although it might be of importance for the secretion of fluid and proteins. Using a new method with calcium-selective disc electrodes, which gives an easy and exact estimation of ionized calcium, the Ca²⁺ activity in pilocarpine-stimulated parotid saliva of 10 healthy males was measured. With increasing flow-rate (0.1–0.6 ml/min), the concentration of ionized calcium rose exponentially from 1.13 ± 0.36 mequiv./litre (0.57 ± 0.18 mmol/litre) to 1.76 ± 0.21 mequiv./litre. At higher flow-rates (up to 2.5 ml/min), a plateau was observed. The total calcium concentration and the ionic calcium concentration were closely correlated (r = 0.93) and the ratio ionic/total remained nearly constant at 0.54 at all flow-rates.     

A method for determining concentrations of calcium complexes in human parotid saliva by gel filtration

Saliva was collected at two flow rates (approx. 0.15 ml/min, pH 6.6 and 1.5 ml/min, pH 7.4) from one subject. The saliva was gel-filtrated at 37 °C in a column, equilibrated and eluted with a buffer containing Ca²⁺ at a concentration equal to that of saliva. Three peaks containing the Ca complexes were identified as proteins (I), phosphate, citrate, lactate (II) and bicarbonate (III). In the weakly stimulated saliva, the total Ca was 0.61 mmol/l, distributed as Ca²⁺ (45 per cent), bound to proteins (10 per cent), complexes to the inorganic ions (35 per cent) and to the organic acids (10 per cent). For strongly stimulated saliva (total Ca 1.15 mmol/l), the corresponding figures were 43.5, 8.7, 40 and 7.8 per cent respectively. The higher total Ca concentration in the strongly stimulated saliva was recovered mainly as Ca²⁺ and Ca complexed to carbonate.     

Isolation and characterization of the basic proline-rich proteins from rat parotid saliva

Five fractions of basic proline-rich proteins were isolated from rat parotid saliva, obtained by surgical cannulation of the ducts. The purification procedures employed DEAE-Sephadex to isolate a heterogeneous break-through fraction containing the basic proline-rich proteins, followed by gel filtration on Sephadex G-200 to separate the high molecular weight glycoprotein, fraction A, from the other basic proline-rich proteins which were resolved into four additional fractions, SP-1 to SP-4, by ion exchange chromatography on SP-Sephadex. The proteins differed in their amino acid composition and content of neutral and amino sugars. All the proteins were characterized by a high proportion of proline (approx. 40 mol per cent) and glycine (11–23 mol per cent). Four of the fractions were also enriched in glutamic $\text{acid}\text{glutamine}$ (19–26 mol per cent). The exception was fraction SP-4, which contained lower levels of glutamic $\text{acid}\text{glutamine}$ and has no counterpart in human basic proline-rich proteins. Fraction A, the basic glycoprotein, was heavily glycosylated (59 mol per cent), whereas SP-2 and SP-4 were less glycosylated. Fractions SP-1 and SP-3 contained low levels of neutral and amino sugars. Basic proline-rich proteins constitute a smaller percentage of the total protein in rat parotid saliva than they do in human parotid saliva (10.5 versus 40 per cent). Rat basic glycoprotein fraction constitutes less than 1 per cent whereas the human glycoprotein fraction constitutes 17 per cent. Rat basic proline-rich proteins appear to be larger and less basic than most of the human basic proteins, and they resolve into fewer protein fractions (4 versus 9) with SP-Sephadex chromatography.     

Circadian rhythms in the flow rate and proportional contribution of parotid to whole saliva volume in man

Seven subjects collected both left-parotid saliva and the remaining whole saliva simultaneously under resting conditions and under“sour-lemon-drop” (SLD) stimulation at five different times each day for spans of 11–17 consecutive days. Leastsquares cosine analysis of the data demonstrated statistically significant circadian rhythms under resting conditions in the estimated flow rates from both parotid glands (mean level, 0.13 ± 0.02 ml/min; amplitude, 0.05± 0.01 ml/min; acrophase, ?234.1 ± 5.0 ° [i.e. 3.36 p.m.± 20min.]), and in the flow rate of whole saliva (mean level, 0.46 ±0.05 ml/min; amplitude, 0.15 ± 0.03 ml/min; acrophase, ?250.2 ± 5.9 °) as well as in the parotid contribution to whole saliva volume (mean level, 28.4± 3.6 per cent; amplitude, 3.5±0.4 per cent; acrophase, ?165.9± 19.2 °). Under SLD-stimulation in which the left parotid flow rate was maintained constant at 1 ml/min, statistically significant circadian rhythms could not be demonstrated in the flow rate of whole saliva or in the proportional contribution of parotid to whole saliva.     

Differences in basic proline-rich proteins in rat parotid saliva following chronic isoproterenol treatment or maintenance on a liquid diet

The basic proline-rich proteins (BPRP) in the stimulated parotid saliva of rats treated for 8 days with isoproterenol and rats fed a liquid diet for 2 weeks were compared to those in the stimulated parotid saliva of untreated rats fed a stock pelleted-diet (control). In the control, the BPRP were separated into 5 groups designated Peak A (the basic proline-rich glycoprotein), SP-1, SP-2, SP-3 and SP-4. The percentage of BPRP in each group was as follows: Peak A, 6.5 per cent; SP-1, 37 per cent; SP-2, 6.5 per cent; SP-3, 32.4 per cent; SP-4, 17.6 per cent. In the parotid saliva of rats fed the liquid diet, proteins corresponding to Peak A and SP-2 were not present, the proportion of BPRP in SP-4 was increased to almost 90 per cent while the proportions of material in SP-1 and SP-3 were reduced to 3 and 8 per cent, respectively. In the saliva of rats subjected to chronic isoproterenol treatment, a protein corresponding to SP-4 was not present; proteins corresponding to Peak A, SP-1 and SP-3 were present and in amounts similar to their proportion in untreated rats although material in SP-2 increased to 36 per cent.     

Immuno-electrophoretic and chemical analyses of human parotid saliva

Dialyzed and lyophilized parotid saliva, collected from a single donor, was characterized with respect to its chemical composition and number of components. The number of components was determined by immuno-electrophoretic analysis, and these results compared with those obtained by zone electrophoresis in polyacrylamide. The carbohydrate content of parotid saliva consisted of 3.5 per cent fucose, 3.4 per cent N-acetylglucosamine, 3.1 per cent mannose, 2.6 per cent galactose, and 0.43 per cent sialic acid. Protein comprised 61.4–63.5 per cent of the total composition. Proline, glycine, and glutamic acid accounted for 65 per cent of the amino acid residues. Flatbed polyacrylamide electrophoresis revealed 17–18 bands. Immunoelectrophoresis using anti-parotid saliva sera revealed at least 12 antigens. Six of these were reactive with an anti-submaxillary saliva serum, while three were reactive with an anti-human serum. Fractionation of parotid saliva by DEAE-cellulose chromatography, separated the parotid antigens into reaction mixtures, which gave less complex patterns when examined by immunoelectrophoresis. Only one additional antigen was revealed that was not detected in the unfractionated secretion. The precipitin arcs of amylase, IgA, albumin, and a parotid glycoprotein were identified. Three other uncharacterized antigens were shown to share antigenic determinants with the parotid glycoprotein. The major advantage of immuno-electrophoresis was its ability to differentiate antigenically related components in complex mixtures.     

Nerve growth factor concentration in human saliva

OBJECTIVE: The aims of this study were to measure the normal concentration of nerve growth factor (NGF) in healthy human saliva and to investigate the effects of age and gender differences on saliva NGF level. MATERIALS AND METHODS: Resting whole, stimulated parotid, and stimulated submandibular/sublingual saliva were collected from 127 healthy volunteers with ages ranging from 20 to 81 years. The saliva NGF concentration was measured by enzyme immunoassay. RESULTS AND CONCLUSIONS: The mean concentrations of NGF were 901.4 +/- 75.6 pg ml(-1) in resting whole saliva, 885.9 +/- 79.9 pg ml(-1) in stimulated parotid saliva, and 1066.1 +/- 88.1 pg ml(-1) in stimulated submandibular/sublingual saliva. The stimulated submandibular saliva showed lower NGF concentrations with increasing age (rho = -0.296, P = 0.001). The NGF concentrations of resting whole saliva (P = 0.025) and stimulated parotid saliva (P = 0.005) were significantly higher in women than men. The NGF concentration of stimulated submandibular saliva was significantly higher than stimulated parotid saliva (P = 0.005) and significantly correlated with stimulated parotid saliva NGF level (rho = -0.244, P = 0.008). We found measurable concentrations of NGF in all three sources of saliva; the concentration was affected by the source for the stimulated parotid and submandibular saliva, age for stimulated submandibular saliva, and gender difference for resting whole saliva and stimulated parotid saliva.     

The basic proline-rich proteins in human parotid saliva from a single subject.

Eleven Chromatographic fractions of basic proline-rich proteins isolated from parotid saliva from a single donor showed similar but not identical chemical compositions; proline, glycine and glutamic acid-glutamine accounted for 70–80 per cent of the total residues in the proteins. The basic amino acids, lysine and arginine, accounted for a further 10 per cent. All the proteins were in freshly-collected saliva and did not result from post-secretory proteolysis. The most basic of the proline-rich proteins, I-B9, was purified and characterized. The amino terminal residue was serine and the carboxyl terminal residue arginine. The molecular weight determined by quantitative end-group analyses was 9000–9500. Protein I-B9 was resistant to hydrolysis by collagenase and Staphylococcus aureus protease, whereas papain and subtilisin extensively degraded it. I-B9 fragments from clostripain and elastase digestions were isolated for use in sequence determination. Basic proline-rich proteins, acidic proline-rich proteins and proline-rich glycoprotein accounted for 23, 30 and 17 per cent, respectively of the total protein in the parotid saliva.     

Biochemical aspects of calculus formation

In an effort to identify factors responsible for individual variations in the amount and rate of salivary calculus formation, submaxillary and parotid secretions were examined from twenty heavy and twenty light calculus formers. The samples were assayed for total calcium, magnesium and phosphorus and for lysozyme and acid phosphatase activity. Individual and pooled submaxillary and parotid samples from five heavy and five light calculus formers were compared by polyacrylamide disc electrophoresis, immunoelectrophoresis, immunodiffusion and adsorption on hydroxyapatite. Reciprocal adsorption was also carried out on antisera to the submaxillary saliva from the two clinical groups. The mean calcium concentration of submaxillary saliva was significantly higher in heavy calculus formers than light formers. In the group of heavy formers, 75% of the values were above 10 mg%; in light formers, 20%. There were no significant differences in submaxillary magnesium and phosphorus or parotid calcium and phosphorus. Lysozyme activity, but not acid phosphatase was significantly lower in heavy formers, suggesting that alteration of bacterial cell walls can affect calculus formation. The electrophoretic and immunochemical studies strongly suggest that there are no proteins present in saliva of heavy calculus formers that are absent in light formers. There were, however, differences in quantities of the various salivary proteins in different individuals. Although no quantitative differences characteristic of heavy or light calculus formers as a group could be discerned, variation was sufficient to warrant further quantitative study.     

The effect of oral contraceptives on the concentration of some salivary substances in women.

Samples of stimulated whole saliva, as well as individual parotid and submandibular gland secretions, were collected repeatedly from twelve 23–26 years old women during one or two menstrual cycles before administration of oral contraceptives (Follinyl^®, Recip, Sweden; norgestrel 0.5 mg and ethinyloestradiole 0.05 mg) and for about 2 months after the start of the hormone administration.Although large individual variations in concentration of various substances were found in response to the hormone administration, some of the parameters showed systematic and significant changes. In stimulated whole saliva, protein, sialic acid, hexosamine, fucose, hydrogen ion concentration and total electrolyte concentration decreased. The secretion rates for both parotid and submandibular secretions increased. The sodium and hydrogen ion concentrations increased in parotid secretion and sodium in submandibular secretion. The differences in concentration could not be explained by differences in secretion rate.Variations in concentrations were for some parameters smaller during the hormone period than during the control period, indicating a stabilizing effect of the hormones.     

Spontaneous Ca2+ Oscillations in Rat Parotid Ducts Are Associated with Cell Swelling

Imaging analysis using multiphoton microscopy revealed that rat parotid ductal cells exhibit spontaneous Ca²⁺ oscillations in the absence of calcium mobilizing agonist stimulation. This spontaneous Ca²⁺ release was first observed during the monitoring of Ca²⁺ transients during continuous perfusion at 37°C, and the protocol for cell preparation was modified to allow consistent observation of spontaneous oscillations. Spontaneous Ca²⁺ oscillations were completely blocked by application of the purinergic receptor inhibitors PPADS and suramin. Simultaneous observation of fura-2 fluorescence and differential interference contrast (DIC) images showed that spontaneous elevations in intracellular Ca²⁺ concentration ( [Ca²⁺] _i) were well correlated with changes in the shape of ductal cells. Using a plasma membrane fluorescence probe we found that the changes in DIC images reflected spontaneous cell swelling of ductal cells. The present findings suggest the possibility that purinergic receptors mediate spontaneous Ca²⁺ oscillations in parotid ductal cells and regulate electrolyte reabsorption from the primary saliva under resting conditions. Cell swelling concomitant with a spontaneous increase in [Ca²⁺] _i was an unexpected result because an agonist-induced increase in [Ca²⁺] _i has been shown to induce cell shrinkage in ductal cells. When spontaneous Ca²⁺ release was compared to the carbachol-induced Ca²⁺ response, there were significant differences in the speed of Ca²⁺ elevation and duration of the Ca²⁺ response. Our data suggest that the different patterns of Ca²⁺ responses in parotid ducts might activate different ion channels and/or ion transporters and cause opposite cell shape changes.     

Salivary secretion and dental caries experience in drug addicts

The flow rate and pH of parotid saliva, the total concentration of salivary proteins, inorganic phosphate, calcium and amylase and the DMF index were studied in young males making improper use of drugs. Patients were classified in the following main groups: (A) with predominance of amphetamines; that is, this drug was taken most frequently and in greatest dosage during the last year; (M) with predominance of marijuana; (AM) with predominance of amphetamine and marijuana; (N) without predominance. In patients belonging to groups A and AM, parotid salivary flow induced after stimulation by intra-oral citric acid was reduced to 26.2 and 41.1 per cent, respectively, of the values obtained in healthy control subjects. Total protein, phosphate concentration and the pH of the saliva were significantly decreased; on the other hand, calcium concentration showed a remarkable increase in the saliva of amphetamine-addicted patients. However, the product calcium × phosphate was significantly decreased. The caries index of patients belonging to groups A and AM was 74 and 90 per cent higher, respectively, compared with control values. In group M, parotid salivary flow rate and most of the parameters studied did not differ from those of the controls. The average caries index of those patients was only 28 per cent higher than the controls.     

Hyaluronan (hyaluronic acid) and its regulation in human saliva by hyaluronidase and its inhibitors

The presence of hyaluronan (HA), hyaluronidase and hyaluronidase inhibitors has been assayed in pure resting and stimulated parotid saliva and also in resting and stimulated mixed saliva utilizing an ELISA-type assay and its modifications. Results confirmed the presence of hyaluronan in all saliva specimens which generally decreased upon stimulation. Hyaluronan in parotid saliva was of high molecular weight (> 200,000 kDa) whilst that in whole saliva in the floor of the mouth had a molecular weight between 20,000 kDa and 200,000 kDa, presumably because of cleavage by bacterial hyaluronidases. Hyaluronidase detection was variable in saliva, being present in some specimens of unstimulated parotid saliva, but in fewer specimens of stimulated saliva. Hyaluronidase was detected in parotid and whole saliva, both in the resting and stimulated state, at pH 3.7. Unstimulated whole saliva also showed hyaluronidase activity at pH 6.8, suggesting a different origin for this hyaluronidase. Hyaluronidase inhibitors were identified in both parotid and mixed whole saliva. There was an inverse relationship between the presence of hyaluranidase and the presence of hyluronidase inhibitors, suggesting a feedback mechanism. The possible significance, interactions and function of hyaluronan, hyaluronidase and its regulation by hyaluronidase inhibitors in saliva is discussed, particularly in relation to intra-oral wound healing and periodontal disease.     

Reduction of carbonic anhydrase activity in the submandibular salivary glands of zinc-deficient rats

Male weanling rats fed a diet containing 0.4 parts/10⁶ zinc for 4 weeks exhibited a 26 per cent reduction in carbonic anhydrase (CA) activity in the submandibular gland compared with pair-fed controls. No reduction in CA activity was evident in the parotid gland, kidney or pyloric portion of the stomach. CA activity of whole blood was the same in both groups. Correction for CA activity of blood did not change the ratios of experimental to control CA activity in submandibular gland and stomach and increased the ratio in kidney. Alkaline phosphatase activity was decreased by 44 per cent in the submandibular gland and 41 per cent in the parotid gland of the zinc-deficient group, confirming earlier reports. Reduction in buffering capacity and changes in the pH of saliva may contribute to the poor food intake of the zinc-deficient rats.     

Saturation of human saliva with respect to calcium salts

It may be assumed that free ionic concentrations of calcium and phosphate in resting saliva tend to equilibrate with those in plaque fluid, and that salivary data can therefore be used to illustrate chemical conditions in both saliva and plaque. In the present study, salivary data collected from the literature or obtained in our laboratory were used to calculate degrees of super- and undersaturation with respect to apatites, brushite, beta-tricalcium phosphate, octacalcium phosphate, calcium carbonate and calcium fluoride in the pH range from 3 to 9. Concentrations of calcium, phosphate, fluoride, carbonate, and background ion strength of resting parotid saliva, resting submandibular saliva, and resting and stimulated whole saliva were entered into a computer program, and curves illustrating saturation in the pH range 3-9 constructed. It was found that oral fluids are supersaturated with respect to apatites above pH 5.3 and with respect to octacalcium phosphate and beta-tricalcium phosphate above pH 6. Parotid saliva was undersaturated with respect to brushite whilst submandibular saliva was supersaturated with respect to that salt in the pH range 6-8. Stimulated whole saliva with 25 mmol/l carbonate became supersaturated with respect to calcium carbonate only above pH 7.3, which may explain the absence of this salt in the human oral cavity. To maintain the saturation of oral fluids with respect to calcium fluoride, i.e. to ensure its survival in the mouth required 6 ppm fluoride in the aqueous phase. Therefore, this salt, the outcome of topical fluoride therapy, will inevitably dissolve in the oral fluids.     

The influence of moderate reduction in dietary sodium on human salivary sodium concentration

Twenty-four healthy subjects were placed for 12–13 weeks on diets that reduced average sodium intake from 145 to 74 m-equiv. Na⁺/day as determined by multiple 24-h urine collections before and during the diet. Whole-mouth resting and stimulated saliva was collected and analysed for flow rate and sodium concentration several times before and during the low-sodium period. Sodium restriction did not influence salivary flow rates but salivary sodium levels fell 25 per cent for resting and 17 per cent for stimulated saliva. Thus moderate reductions in sodium intake are accompanied by significantly lower salivary sodium levels.     

Ca-ATPase activity in salivary glands of rats treated with reserpine,isoproterenol, terbutaline or dobutamine

Ca-ATPase activity (mol P_i/mg protein per min) of submandibular and parotid glands after injection of single or multiple (over seven days) 0.5 mg/kg doses of reserpine was the same as in untreated glands. Twice daily doses (50 mg/kg body wt) of the non-selective β-adrenergic agonist, isoproterenol, for six days, increased Ca-ATPase specific activity of parotid gland by 17 per cent but that of submandibular gland was the same as controls; with dobutamine, the same dosage caused a 53 per cent decrease in submandibular activity and a 31 per cent decrease in parotid. The activity of the entire parotid gland was markedly increased by all three β-agonists, and this was generally a reflection of the induced increase in gland size. The submandibular gland had an increase in total Ca-ATPase activity only with isoproterenol. As gland weight did not change after reserpine, total glandular Ca-ATPase activity was also not altered by it. Thus, calcium accumulation and reduction in Ca-ATPase activity are not necessarily related. However, the dobutamine-induced decrease in Ca-ATPase activity of both glands suggests that there is a β₁-mediated regulation of this enzyme.     

Effects of electrical stimulation of the sympathetic nerve on the levels of β-adrenergic and cholinergic muscarinic receptors and cyclic nucleotides in rat salivary glands

After 30 and 60 min of stimulation, there were decreases of 16 to 19 per cent in β-adrenoceptors in rat submandibular and parotid glands; a 10-min stimulation caused no change. Pre-incubation of the reaction mixtures (stimulated glands) with atenolol, a β₁-antagonist, prevented most dihydroalprenolol (DHP) binding, but with butoxamine, a β₂-antagonist, DHP binding was nearly complete. Thus the β-receptor was of the β₁-subtype. Muscarinic receptors of parotid gland showed no change after 10 min stimulation; after 30 min there was an increase of 12 per cent, and after 60 min, of 28 per cent. With submandibular gland, there was also no change at 10 min but, at 30 min, there was a 25 per cent increase, and at 60 min, a decrease of 18 per cent. Cyclic-AMP levels of parotid gland were markedly elevated after 10 min of stimulation (9-fold increase above controls) but, at 30 and 60 min, there was only a 1.6-fold increase. In submandibular gland, cyclic-AMP increased 10-fold at 10 min; at 30 min it was 2.5 times control levels and at 60 min, 1.9 times. Cyclic-GMP levels of parotid gland increased 34-fold after 30 or 60 min of nerve stimulation, but only 1.6-fold at 10 min. With submandibular gland, there was a 22-fold increase at 10 min, but at 30 and 60 min this was 15- and 12-fold, respectively. Thus β-adrenergic and muscarinic receptor densities and levels of cyclic AMP and CMP change after acute stimulation of the sympathetic nerve to the rat salivary glands; increases in levels of both nucleotides precede the changes in receptor number.     

The bicarbonate concentration in human saliva does not exceed the plasma level under normal physiological conditions

Depending on the secretion rate and nature of the stimulus applied, the bicarbonate concentration ([HCO₃ ^–]) in human saliva has been shown to vary from 1 to 60 mM, with the highest values obtained in secretions from the parotid and submandibular glands. We conducted the present study on five healthy young males, in order to determine whether human saliva [HCO₃ ^–] can exceed the plasma level under normal physiological conditions, and applied a strategy in which we measured the secretory response to various stimulus intensities. Whole saliva stimulation was initiated by chewing paraffin (50–100 chewing cycles/min), parotid saliva stimulation by citric acid taste (0.2–4% citric acid solutions), and both secretions were also collected during systemic medication using pilocarpine (5 mg). Our results showed that the parotid and whole saliva flow rates were closely correlated to the intensity of the secretion-inducing stimulus applied (taste, chewing, and pilocarpine). This was also the case for saliva [HCO₃ ^–], all of which was present in the form of physically dissolved CO₂, H₂CO₃, and HCO₃ ^–. The highest mean [HCO₃ ^–] (i.e. 37.75 mM) was found in parotid saliva after stimulation by pilocarpine in combination with 4% citric acid taste. Under normal physiological conditions (i.e. without pilocarpine) the saliva [HCO₃ ^–] was similar to or below the plasma level in both secretions. Received: 21 March 2000 / Accepted: 7 June 2000