The Effect of Long-Term Antihypertensive Therapy on the Change in Secretion and Calcium, Bicarbonate and Phosphate Ion Concentration in Non-Stimulated and Stimulated Saliva.

OBJECTIVE: The aim of this study was to evaluate the amount of saliva secreted and calcium, bicarbonate, and phosphate ion concentration in patients receiving antihypertensive for five years or over five years (patient group) and in healthy patients (control group).
MATERIAL AND METHODS: The patient or experimental group included 31 subjects who were admitted to a cardiovascular clinic and had been receiving an antihypertensive drug therapy for more than five years. The control group included 31 healthy subjects. The measured amount of saliva was further used to determine the calcium, phosphate and bicarbonate ion concentration values. Calcium and phosphate ions were determined spectrophotometrically, while bicarbonate ions were determined by titration.
RESULTS: A two-way-test (Student's test) was used to compare the values of variables. The amount of excreted saliva was statistically significantly lower in the patient group in non-stimulated (1.739 mL/5 min) and stimulated saliva (3.594 mL/5 min). Calcium ion concentration was statistically significantly lower in patient group in resting saliva (6.143 mg/dL). Bicarbonate and phosphate ion concentration in patient group was statistically significantly higher in non-stimulated (bicarbonate ion = 14.041 mmol/L, phosphate ion = 2.818 μmol/L) and stimulated saliva (bicarbonate ion = 10.872 mmol/L, phosphate ion = 1.454 μmol/L), respectively.
CONCLUSION: A reduced amount of saliva and calcium ion concentration indicates the possibility of a higher frequency of hard dental tissue demineralization process. On the contrary, the increase in the phosphate and bicarbonate ion concentration in the patient group affects the regulation of acid-base balance, thus having a preventive effect.

Introduction

Saliva has several different types of functions (antibacterial properties, digestion, cleansing, buffering, lubrication, etc.,) which play an important role in maintaining both oral and overall health of the body (, ). Saliva secretion is controlled by three basic centers: primary (localized in the medulla oblongata), secondary (thalamus), and tertiary salivation center (opercula- insular zone of the cerebral cortex) (). Based on the manner of production and excretion, mixed saliva is divided into stimulated saliva and resting saliva. Stimulated mixed saliva occurs as a result of the effect of the most diverse factors, which act directly in the oral environment on numerous and diverse receptors, and/or indirectly through the sense of sight, hearing or smell, thus causing its secretion to be enhanced. Resting mixed saliva is produced as a secretion product of the entire glandular apparatus of the oral cavity in conditions when there is no external stimulus for its creation.

The acidity of the oral cavity environment is closely related to the amount and the chemical composition of the secreted mixed saliva. The pH measurement of mixed saliva has shown a wide range from the most acidic (pH=6.1) to alkaline (pH=7.8). Acidity of saliva is also associated with the measurement period (during day or night), as well as with the volume of salivary secretion, i.e. whether it is stimulated or resting salivation and it is important in patients undergoing radiotherapy (, ).

Bicarbonate buffer is excreted in enhanced concentration during stimulated salivation when it represents the dominant buffer. Due to the multiplier effect of enhanced concentration of this buffer, mixed saliva becomes alkaline, reaching a pH of 7.8. In such alkalization conditions, and due to the effect on the acid products present in saliva, it can be considered as a precautionary measure in preventing demineralization of the hard-dental tissues’ surface (, ).

Phosphate buffer is the basis of the buffer system in resting saliva. It is extremely important in the physiological processes of resting saliva (during 22 hours in a day), and in the period when there is no stimulated saliva secretion. For remineralization processes of the surfaces of hard-dental tissues, apart from the buffer, the amount of calcium and phosphate ions present in the saliva is pivotal (, ). A high concentration of these ions in saliva prevents the demineralizing effect of water while remineralizing the enamel’s damaged surface at the same time.

Different local and systemic diseases may be the reason for qualitative and quantitative changes in salivary secretion (Parkinson’s disease, Alzheimer’s disease, glandular infections, tumors and use of certain drugs) (, ). The drugs that most commonly cause problems in the qualitative and quantitative secretion of saliva belong to different groups such as antihypertensives, antihistamines, opioids, and antidepressants (, ).

Antihypertensives may be divided into two broad groups: the first group of drugs which directly or indirectly block the renin-angiotensin system (the predominant effect is to cause vasodilatation) and the second group of drugs works by increasing water and sodium excretion (diuretics and calcium channel blockers). Different groups of drugs (i.e. inhibitors of the angiotensin converting enzyme, angiotensin receptor blockers, calcium channel blockers, thiazide diuretics), in addition to the antihypertensive effect, can also have an effect on other systemic parts of the human body, as well as on the qualitative and quantitative properties of the stimulated and non-stimulated saliva ().

This study aimed to determine changes in the amount of saliva excreted and calcium, bicarbonate and phosphate ion concentration in stimulated and resting saliva in patients on long-term antihypertensive therapy (longer than five years), and to compare them with findings in a healthy population.

Material and methods

The study included 62 subjects divided into two groups: patient and control. The patient or experimental group included 31 (18 males, 13 females) subjects who were admitted to a cardiovascular clinic and who had been receiving antihypertensive drug therapy for more than five years. Twelve patients used a combination of beta-adrenergic blockers with ACE inhibitors, ten patients were on beta-adrenergic blockers and diuretics, while the remaining nine patients used a calcium antagonist in therapy of hypertension. The control group included 31 healthy subjects (13 males, 18 females). The study was conducted at the Clinic center and the Biological laboratory of the Faculty of Natural Sciences and Mathematics University of Banja Luka.

The patient group inclusion criteria were as follows: patients diagnosed with hypertension, patients who had been only on antihypertensive therapy for five years or more, hospitalized patients under medical supervision, patients without disease or clinical condition that could affect the qualitative and quantitative characteristics of salivation.

The patient group exclusion criteria were as follows: patients with occasional manifestations of hypertension, patients receiving the antihypertensive therapy occasionally or those receiving it less than five years, patients with disease or clinical condition that could affect the qualitative and quantitative characteristics of salivation.

The control group inclusion criteria were as follows: healthy patients with physiological pressure; patients who were not receiving any therapies..

The control group exclusion criteria were as follows: patients taking occasional antihypertensive drugs or any therapy that may affect the parameters being evaluated.

Each of the subjects received an information form, based on which they voluntarily consented for participation. The study was approved by the Ethics Committee of the Clinic center in Banja Luka, under the no. 01-5-355.2/12, and adhered to the principles outlined in the Declaration of Helsinki.

Sample collection

After the health history had been taken and processed, the extraoral and intraoral examinations were completed, and the resting and stimulated saliva sampling was done.

Non-stimulated and stimulated saliva samples of the patient were collected before breakfast, without taking any food or drinks previously, and without oral hygiene between 6.30 a.m. and 7.30 a.m. While preparing to take a sample, the patient was seated in a chair with the head slightly tilted forward, with relaxed arms and shoulders.

Non-stimulated saliva was collected in the mouth for five minutes, spat into a sterile measuring cylinder and closed. Each sample was marked with the letter N (non-stimulated, i.e. resting) and identified by an ordinal number from 1 to 31.

Stimulated saliva was collected from patients after they had been chewing a paraffin ball (8 mm in diameter) for 5 minutes, thus stimulating saliva secretion. The saliva obtained by stimulation was spat into a sterile measuring cylinder and closed. The samples obtained were marked with numbers from 1 to 31 and with letter S (stimulated) for each patient.

During their transport to the laboratory (10 minutes), the samples were stored in a mobile refrigerator at a temperature of 4 °C until analyzed. The saliva sample obtained was poured into a glass measuring cylinder (graduated by 1 ml).

Sample analysis

The measured amount of saliva was recorded and divided by 5 to obtain the value of a milliliter of saliva per minute, and the final result was recorded in the questionnaire prepared. The measured amount of saliva was further used to determine the calcium, phosphate and bicarbonate ion concentration values. Calcium and phosphate ions were determined spectrophotometrically, while bicarbonate ions were determined by titration.

Calcium ion concentration was determined by the reaction of saliva sample and calcium arsenazo III reagent (2,2´-[(1,8-Dyhydroxy-3,6-disulfonaphthylene-2,7-bisazo)] bisbenzenearsonic acid), 2,7-Bis (2-arsonophenylazo) chromotropic acid, in such a way that 1ml arsenazo III reagent was added to 10 μL of saliva, mixed and left for 3 minutes at room temperature (20-25 °C). After that, spectrophotometric measurement was performed with UV -1800 Shimadzu Spectrophotometer (Shimadzu Corporation, Kyoto, Japan) at 650 nm wavelength and obtained values were read on a spectrophotometer. The color intensity was directly proportional to calcium concentration in the saliva sample. The obtained values were entered in a formula ():

calcium concentration = SA Abs/ST Abs x10 = mg calcium/dL.

sample absorbance x 10

standard absorbance

SA Abs – sample absorbance

ST Abs – standard absorbance

The obtained values were tabulated.

The phosphate concentration was determined by molybdenum reaction by Goldenberg and Fernandez’s spectrophotometric method, modified by Bardow et al. (). Buffer (reaction mixture consisting of 10% trichloroacetic acid (TCA), 1% urea and 3% Mohr’s salt) was added to saliva. The buffer was prepared directly in the laboratory. After 10 minutes, centrifugation was performed (Tehtnica Centric 200R) at 5000 rpm. It separates the supernatant and adds concentrated sulfuric acid and 4.5% ammonium molybdate to the supernatant. The preparation was kept for 20 minutes at room temperature and then the absorbance was spectrophotometrically measured at 700 nm. The standard curve was made in the range from 0 to 10 µmol / L of phosphate.

Bicarbonate concentration is determined by titration with 0.1M HCl ranging from pH 7 to pH 3, by adding HCl to the sample in a volume of 100 µL. Titrated saliva was measured in the said solution and each value was recorded. After the measurement, it was entered into the formula and final value was obtained. The formula used for calculating bicarbonate was:

C1V1=C2V2

C1 - acid concentration

C2 – the unknown value that we are looking for,

V1 – total titration agent volume,

V2 – total volume (saliva and titration agent)

We divided the obtained C2 value by ΔpH, which represents the difference between the first pH sample and the last pH sample measured. Values were expressed in mmol/L. All values obtained from saliva samples were documented in the questionnaire for each patient individually.

Statistical analysis

A two-way-test (Student's test) was used to compare the values of the variables under the following assumptions: the variances of the compared data groups are unequal and the difference of the mean values of the variables being compared is equal to zero. The differences were considered significant at P<0.05. Standard descriptive statistics tools (frequency tables, grouping of data into classes, graphs, calculation of arithmetic mean and minimum and maximum values, standard deviation and variance) were used to display and analyze the data obtained by the research.

Results

The values obtained by measuring the amount of saliva and calcium, phosphate and bicarbonate ion concentration in patient and control subjects in stimulated and resting saliva are presented in Tables 1-4 and Figures 1-4.

Table 1

Amount of excreted saliva.

	Non-stimulated saliva		Stimulated saliva
Saliva amount (ml/5 min)	Experimental (N)	Control          (N)	Experimental (S)	Control (S)
Total number	31	31	31	31
Average value	1.739	3.535	3.594	6.271
Min value	0.400	1.000	0.500	2.200
Max value	5.800	8.300	11.000	11.200
St. deviation	1.30810	1.82365	2.73982	2.63947
Variation coefficient	75.23%	51.58%	76.24%	42.097%
t value	0.000108		0.00048

Table 2

Calcium ion concentration value in stimulated and non-stimulated saliva excreted.

	Non-stimulated saliva		Stimulated saliva
Ca (mg/dl)	Experimental (N)	Control (N)	Experimental (S)	Control (S)
Total number	31	31	31	31
Average value	6.143	7.922	5.911	6.541
Min value	0.917	0.464	1.409	3.217
Max value	14.173	13.805	12.987	12.481
St. deviation	3.53591	2.87808	3.10942	2.11706
Variation coefficient	57.56%	36.33%	52.60%	32.37%
t value	0.033934		0.355793

Table 3

Phosphate ion concentration value in stimulated and non-stimulated saliva.

	Non-stimulated saliva		Stimulated saliva
Phosphates (μmol/l)	Experimental          (N)	Control          (N)	Experimental          (S)	Control          (S)
Total number	31	31	31	31
Average value	2.818	1.388	1.454	0.565
Min value	0.109	0.054	0.018	0.018
Max value	17.636	3.254	5.927	3.690
St. deviation	3.51657	1.07435	1.47937	0.77418
Variation coefficient	124.79%	77.41%	101.77%	137.02%
t value	0.037135		0.004839

Table 4

Bicarbonate ion concentration value in stimulated and non-stimulated saliva.

	Non-stimulated saliva		Stimulated saliva
Bicarbonates (mmol/l)	Experimental          (N)	Control          (N)	Experimental (S)	Control          (S)
Total number	31	31	31	31
Average value	14.041	9.929	10.872	5.964
Min value	2.939	2.890	2.562	2.467
Max value	37.131	29.600	38.300	10.219
St. Deviation	9.48100	5.07875	9.41473	2.47299
Variation coefficient	67.53%	51.15%	86.59%	41.47%
t value	0.03870		0.00819

Figure 1

The average amount of non-stimulated and stimulated saliva in the experimental and control group (E- experimental group, C- control group).

Figure 2

The average calcium concentration value in non-stimulated and stimulated saliva in the experimental and control group (E- experimental group, C- control group).

Figure 3

The average phosphate concentration value in non-stimulated and stimulated saliva in experimental and control group (E- experimental group, C- control group).

Figure 4

The average bicarbonate ion concentration value in non-stimulated and stimulated saliva in experimental and control group (E- experimental group, C- control group).

The average amount of saliva of the stimulated and resting saliva in patient and control subjects is presented in Table 1 and Figure 1. The results obtained show a statistically significant difference in the amount of non-stimulated saliva excreted between the subjects from the patient (1.739 mL/5 min) and control groups (3.535 mL/ 5min) (p: 0.00018, p<0.05), as well as in stimulated saliva where there is a statistically significant difference between the two groups (3.594 mL/5min / 6.271 mL/5min) (p: 0.00048, p<0.05).

The calcium concentration values in stimulated and non-stimulated saliva in patient and control groups are presented in Table 2 and Figure 2. In non-stimulated saliva, there is a statistically significant difference between the average values of patient (6.143 mg/dL) and control subjects (7.922 mg/dL) (p: 0.03, p<0.05), while there is no statistically significant difference between the two groups in stimulated saliva (5.911 mg/dL / 6.541 mg/dL) (p: 0.35, p>0.05).

The average phosphate concentration values in non-stimulated and stimulated saliva in patient and control subjects are presented in Table 3 and Figure 3. In non-stimulated (2.818 μmol/L / 1.388 μmol/L) (p: 0.03, p<0.05) and stimulated saliva (1.454 μmol/L / 0.565 μmol/L) (p: 0.004, p<0.05) there are statistically significant differences in the average values between patient and control groups.

The bicarbonate concentration value in resting and stimulated saliva in patient and control groups is presented in Table 4 and Figure 4. In non-stimulated (14.041 mmol/L / 9.929 mmol/L) (p: 0.03, p<0.05) and in stimulated saliva (10.872 mmol/L / 5.964 mmol/L) (p: 0.008, p<0.05), there are statistically significant differences in the average values between the patient and control group subjects.

Discussion

The qualitative and quantitative features of saliva are related to the biological functions of the entire body and may be related to the emergence and development of both local and several systemic diseases (-). The amount of resting saliva in a healthy person under physiological conditions ranges from 0.3 ml/min to 0.4 mL/min. In the case of reduced saliva secretion (oligosallium), the amount of saliva excreted ranges from 0.2 to 0.4 mL/min, and if the values of saliva excretion are less than 0.2 mL/min that is called hyposalivation (, ).

In this study, a statistically significant difference (p=0.000108) was found in the average values of the resting saliva excreted in the patient group (1.739 mL/ 5 min) compared to the control group (3.535 mL/ 5min). Moreover, a statistically significant difference (p=0.00048) of the average values of stimulated saliva excreted in the patient group (3.594 mL/5 min), compared to the control group (6.271 mL/5 min). These results are consistent with those of Leandro (), who measured the amount of saliva excreted in patients using beta blockers. Kagawa et al. () obtained slightly different results in which there was no difference in the amounts of resting and stimulated saliva excreted in patient and control group. Consistent with our study, Nauntofte and Twetman () reported an increase in xerostomia in patients on diuretic therapy. The reduced amount of resting and stimulated saliva excreted in the patient group may be related to the long-term usage of antihypertensive medications, as confirmed by Murray () in his study.

Skanda () confirmed that there was a close relation between the use of thiazide medication and xerostomia, stating that xerostomia was ten times more enhanced after furosemide ingestion. Cano et al. () suggested that renin-angiotensin system mechanisms complying exclusively vasoconstriction and dilation are not able to completely describe salivary glands local mechanisms on saliva release, since the decrease of angiotensin converting enzyme expression in myoepithelial cells cannot explain lower salivary rates by itself. They considered that local angiotensin converting enzyme, in addition to other cascades, would affect these cells contraction and further reduce saliva flow. In this study, no difference was observed in the administration of certain medication from different groups of antihypertensives in the amount of saliva obtained.

The amount of saliva excreted as well as its physicochemical properties (composition, pH value), with particular emphasis on calcium and phosphate concentrations, are closely related to the emergence of demineralization and carious lesions in the hard-dental tissue area (, ).

Calcium concentration values in saliva, on average, under physiological conditions in a healthy person are 8.8-10.5 mg/dL. This study obtained a statistically significant (p=0.034) average calcium concentration value in resting saliva in patient group (6.143 mg/dL) compared to control group (7.922 mg/dL). Stojšin () obtained approximately the same values of average calcium concentration in resting saliva. The average calcium concentration value in stimulated saliva in patient group was 5.911 mg/dL, while in the control group it was 6.541 mg/dL. Although lower values were obtained in the patient group, they were not statistically significant. By comparing the average calcium concentration values in stimulated and resting saliva, higher values were shown in resting saliva in these two groups. Contrary to the results of this this study, Gauri, Nagarajappa, Bhat () found a lower average value of 5.87 mg/dL in resting saliva, whereas after stimulation, the average calcium concentration value was 7.17 mg/dL. Jarvinen () in his study obtained a lower calcium and phosphate concentration values in resting saliva, which was explained by the small amount of resting saliva in all study groups. Some studies have shown that critical pH value (below 5.5) could be modified if calcium and phosphate substituents were added depending on the amount of saliva excreted and the pH value (, ).

Phosphate buffer is the dominant buffer in resting saliva. It is a combination of primary and secondary phosphate, where the concentration in resting saliva is 7-8 mM/L, and during the salivation stimulation period, this value decreases to 2-3 mM/L ().

The results of the current study showed a statistically significant difference in phosphate concentration value between the patient and control groups, with values higher in resting saliva compared to stimulated saliva. The average phosphate concentration value in resting saliva in the patient group was higher (2.818 μmol / L), compared to the control group (1.388 μmol / L) (p<0.05). In stimulated saliva, phosphate concentration values were higher in the patient group (1.454 μmol / l) than in the control group (0.565 μmol / L). The higher phosphate concentration obtained in the patient group saliva could have an impact on reducing the frequency of demineralization processes and carious lesions on hard dental tissues. Šurdilović () found that phosphate concentration values were significantly high in both stimulated and resting saliva in children with low caries risk. In assessing phosphate concentration results in resting saliva, the potentially possible part that remains bound and neutralized by acidic products in the oral cavity should also be considered (, ).

The physiological values of salivary bicarbonates are between 1-60 mM, with the highest values obtained from the parotid and submandibular gland (). In this study, a statistically significant difference was found between the bicarbonate concentration values obtained in stimulated and resting saliva in both patient and control group. The bicarbonate concentration is higher in resting saliva in both study groups. It amounts to 14.041 mmol/L in the patient group, and 9.929 mmol/L in the control group, while the bicarbonate concentration in stimulated saliva in the patient group is 10.872 mmol/L, and in the control group it is 5.964 mmol/L. During long-term saliva stimulation, although the amount of the excreted saliva increases, the bicarbonate concentration value decreases, with respect to the total amount, thereby depleting its buffering power, regardless of any further stimulation present. An explanation for the low bicarbonate concentration in stimulated saliva, obtained in this study, could be related to the following factors: the sampling of stimulated saliva that was performed in the early morning, where patients did not take any water or food; chewing paraffin without taste that was used as a stimulating agent; and the intensity of the chewing cycles that were done. Bardow et al. () pointed out that the amount of bicarbonate in the saliva depends on the total amount of saliva excreted. In their study, Bardow, Nyvad and Nauntofte () emphasized that bicarbonate concentration in saliva is dependent on the flow rate and on the intensity of the stimulation applied. They also noticed that different values of the obtained concentrations are related to the number of chewing cycles.

Qualitative changes, especially a decrease in calcium ion concentration, can greatly affect possible emergence of demineralization and carious lesions on hard dental tissues. On the contrary, the increased phosphate and bicarbonate ion concentration can stimulate the process of remineralization of hard dental tissues.

Conclusions

With the limitation of this study, it can be concluded that in patients who have been on continuous antihypertensive therapy for five years or more, compared to the healthy population, a reduced salivation with calcium ions deficiency can be expected, which can lead to increased demineralization of hard dental tissues, and contribute to plaque formation and carious lesions. In order to prevent such processes, it is necessary to compensate for the lack of excreted amount of saliva and calcium ions by implementation of effective prevention programs. On the contrary, the increase in the phosphate and bicarbonate ion concentration in the patient group affects the regulation of acid-base balance and remineralization process, i.e. hard dental tissue preservation, and thus has a preventive effect.