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Increased calcium and decreased magnesium and citrate

concentrations of submandibular/sublingual saliva in

sialolithiasis

Yu-xiong Su a,b,1, Kai Zhang b,1, Zun-fu Ke c,1, Guang-sen Zheng a,b, Mei Chu a,b,Gui-qing Liao a,b,*a Department of Oral and Maxillofacial Surgery, Guanghua School of Stomatology, Sun Yat-sen University, 56 Lingyuanxi Road,

Guangzhou, 510055, Chinab Institute of Stomatological Research, Guanghua School of Stomatology, Sun Yat-sen University, 56 Lingyuanxi Road, Guangzhou,

510055, ChinacDepartment of Pathology, Medical School of Sun Yat-sen University, 74 The Second Zhongshan Road, Guangzhou 510080, China

1. Introduction

Obstructive salivary gland disease is one of the most common

problems that afflict salivary glands and is a major cause of

salivary gland dysfunction and sialoadenectomy.1 Sialoliths

located in Whartons duct or in Stensens duct are the most

frequent cause of obstruction andconsequent acute or chronic

infection. The incidence of salivary calculi is 60 cases/million/

year.2 However, the exact cause of sialolithiasis perse remains

elusive.

a r c h i v e s o f o r a l b i o l o g y 5 5 ( 2 0 1 0 ) 1 5 2 0

a r t i c l e i n f o

Article history:

Accepted 11 November 2009

Keywords:

Submandibular gland

Salivary calculi

Saliva

Electrolyte

a b s t r a c t

Objective: The purpose of the present study was to investigate the electrolyte alterations of

submandibular/sublingual saliva in submandibular sialolithiasis patients compared with

saliva from healthy controls.

Design: Submandibular/sublingual saliva was collected from 10 submandibular sialolithia-

sis patients and from 10 sex- and age-matched healthy controls. Ion chromatography was

performed to determine the concentrations of inorganic cations (potassium, calcium,

magnesium, and sodium), inorganic anions (chloride, phosphate, nitrate, and sulphate)and organic anions (thiocyanate, lactate, acetate, and citrate).

Results: The calcium concentration was significantly higher in sialolithiasis patients than in

controls (P < 0.05). In contrast, the levels of magnesium and citrate in the saliva of sialo-

lithiasis patients were significantly reduced compared to the values obtained in controls

(P < 0.05). No significant differences were detected among other ions.

Conclusions: Our findings indicate that saliva electrolyte composition of sialolithiasis

patients is substantially altered with respect to crystallisation mechanisms. Increased

calcium ion as a crystallisation substance and decreased magnesium and citrate ions as

crystallisation inhibitors may be involved in the etiopathology of calculi formation.

# 2009 Elsevier Ltd. All rights reserved.

* Corresponding author at: Department of Oral and Maxillofacial Surgery, Guanghua School of Stomatology, Sun Yat-sen University, 56Lingyuanxi Road, Guangzhou, 510055, China. Tel.: +86 20 83862531; fax: +86 20 83822807.

E-mail address:[email protected](G.-q. Liao).1 The contribution of these authors for the current article was equal.

a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m

journal homepage: http://www.elsevier.com/locate/aob

00039969/$ see front matter # 2009 Elsevier Ltd. All rights reserved.

doi:10.1016/j.archoralbio.2009.11.006
mailto:[email protected]://dx.doi.org/10.1016/j.archoralbio.2009.11.006http://dx.doi.org/10.1016/j.archoralbio.2009.11.006mailto:[email protected]

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Salivary calculi form as a result of mineralisation of debris

that accumulates in the duct.3 Chemically, the composition of

most salivary stones is calcium phosphate in the form of

hydroxyapatite, with small amounts of magnesium, potas-

sium, sodium, ferrum, silicon and chloride.4 Salivary secre-

tions are supersaturated with respect to basic calcium

phosphate salts,5 precipitation of which can contribute to

the formation of sialoliths. Therefore, analysis of salivacomposition in sialolithiasis patients may be enlightening in

the study of calculi formation mechanism.

Whole or mixed saliva collection is easy to perform, but it

maystrikinglybe influenced by gender, age, diet, medications,

alcohol, smoking and systemic diseases such as diabetes

mellitus, asthmas, renal failure and cancer,612 which makes it

unsuitable for estimating glandular physiopathology.13 The

glandular saliva can reflect the pathology of salivary gland

more directly andprecisely. Surprisingly, to date there were no

studies analysing the glandular salivary composition changes

of sialolithiasis patients. Thus, the aim of the present study

was to evaluate the electrolyte alterations of submandibular/

sublingual saliva in submandibular sialolithiasis patientscompared with saliva from healthy controls. Inorganic cations

(potassium, calcium, magnesium, and sodium), inorganic

anions (chloride, phosphate, nitrate, and sulphate) and

organic anions (thiocyanate, lactate, acetate, and citrate) were

analysed with the use of ion chromatography.

2. Materials and methods

2.1. Patients enrollment

A total of 20 subjects from Canton, China, 10 submandibular

sialolithiasis patients (4 men and 6 women) with a medianage of 36 years (range, 2946) and 10 sex- and age-matched

healthy controls, were studied. A medical history of all

patients was taken and a thorough physical examination

was performed. The diagnosis of submandibular sialolithia-

sis was made according to the clinical manifestation and the

imaging examination. The patients were presented with

recurrent episodes of submandibular swelling and pain,

exacerbated by eating. Clinical examination showed mild or

middle swelling of the submandibular area. X-ray radio-

graphy, sonography and/or CT scanning were performed for

imaging diagnosis. All the diagnosis were finally confirmed

by surgical or sialendoscopic removal of sialoliths after

saliva collection. All the patients had no signs of inflamma-tion in the last 2 weeks before saliva collection. The principal

criteria for exclusion included: (a) Suspected Sjogrens

syndrome or any other salivary gland diseases except for

sialolithiasis. (b) Systemic diseases such as cancer, cardio-

vascular diseases, kidney diseases or diabetes. (c) Use of

medications in the past 2 months. (d) Pregnancy or lactation.

(e) A history of smoking. (f) Obese subjects with a body mass

index >24 kg/m2. (g) Periodontal diseases or poor oral

hygiene. (h) With removable dental prosthesis. The criteria

for inclusion of healthy controls included: (a) No history of

salivary gland diseases. (b) Sonography of salivary glands

showed unremarkable results. (c) None of the above

exclusion criteria from (b) to (g). The study protocol was

approved by Institutional Ethic Board, and informed consent

was obtained from all the subjects.

2.2. Saliva collection

The collection was performed in a quiet room under resting

conditions half an hour after toothbrushing with deionised

water. All the saliva samples were collected by a single trainedoperator. The procedures were performed at a fixed time

between 7:30 and 8:30 in the morning after the whole night

fasting in order to minimise fluctuations related to the

circadian rhythm of salivary secretion and composition.

Submandibular/sublingual saliva was collected by syringe

aspiration from the orifices of the Whartons duct which were

separated by cotton rolls in the floor of the mouth, and the

bilateral orifices of the parotid glands were blocked by the

Lashley cups.14 The collection of saliva lasted until 1 ml of

submandibular/sublingual saliva was collected without sti-

mulation. For healthy controls, the time lasted from 2 min to

6 min and 15 s. For sialolithiasis patients, the time lasted from

4 min to 8 min and 20 s. The submandibular/sublingual salivaflowrate wascalculated as ml/min. Allthe salivacollectedwas

free of blood or pus, with a translucent appearance. Saliva

samples were collected in sterile glass bottles on ice and

transported immediately to the laboratory for pre-treatment.

2.3. Saliva pre-treatment

Saliva samples were centrifuged at 14,000 rpm for 20 min at

4 8C in order to remove the contamination elements such as

food debris, bacteria and oral epithelial cells. And then the

supernatant fluid was passed through a 0.45 mm filter

membrane.15 All the samples were stored at 80 8C in glass

containers until analysis. The storage time ranged from 1 to 12weeks.

2.4. Ion chromatography

All the chemicals used during ion chromatography were

analytical reagent grade or better. Distilled deionised water

was used throughout. Ion chromatograph Dionex Dx-600

(Dionex, Sunnyvale, CA, USA) was used for electrolytes

analysis under ion exchange mode. An aliquot of 25 ml sample

was injected for analysis. If necessary, the saliva was diluted

prior to injection. The ion chromatographic protocol was set

up according to the previous description by Chen.15,16

For inorganic cations (potassium, calcium, magnesium,and sodium), the ion chromatography was performed using a

guard column (CG-12A), separator columns (CS-12A) and

suppressed conductivity detection with CSRS-ultra II sup-

pressor, 50 mA. The eluent was 18 mmol/l methylsulphonic

acid at 1.0 ml/min.

For inorganic anions (chloride, phosphate, nitrate, and

sulphate), the ion chromatography was performed using a

guard column (AG-4A), separator columns (AS-4A) and

suppressed conductivity detection with ASRS-ultra suppres-

sor, 50 mA. The eluent was 1.7 mmol/l NaHCO3+ 1.8 mmol/l

Na2CO3+ 100 mg/l C7H5NO at 1.0 ml/min.

For organic anions (thiocyanate, lactate, acetate and

citrate), the ion chromatography was performed using a guard


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column (AG-4A), separator columns (AS-4A) and suppressed

conductivity detection with ASRS-ultra suppressor, 50 mA.

Two eluents were used: 1.6 mmol/l CF3(CF)2COOH + 0.3 mmol/

l NaOH at 1.0 ml/min, forthiocyanate,lactate, andacetate; and

NaOH 10 mmol/l at 1.0 ml/min, for citrate.

2.5. Statistical analysis

The concentrations of saliva ions in sialolithiasis group and in

control group were compared. The submandibular/sublingual

saliva flow rate and the total amount of each saliva ion per

minute were calculated. Data were expressed as median and

quartile range (Q25Q75). The statistical analysis was per-

formedby use of SPSS 14.0software(SPSSInc., Chicago, IL,US).

Because of the limited sample size and the variability of saliva

parameters,17 the non-parametric MannWhitney test was

used. APvalue < 0.05 was considered statistically significant.

3. Results

3.1. Inorganic cations

The median and quartile range of inorganic cations are shown

in Table 1. The median concentrations of potassium and

sodium in sialolithiasis patients were 15.750 mmol/l and

4.335 mmol/l, respectively, showing no significant difference

with what were detected in control group (P= 0.791 and

P= 0.880, respectively). The median calcium concentration in

sialolithiasis patients was 1.035 mmol/l and significantly

higher than that in controls (P= 0.003). In contrast, the level

of magnesium in the saliva of sialolithiasis patients was

0.135 mmol/l, significantly reduced compared to the value

obtained in controls (P= 0.044).

3.2. Inorganic anions

The median and quartile range of inorganic anions are shown

inTable 2. The median concentrations of chloride, phosphate,

nitrate, and sulphate in sialolithiasis patients were

9.095 mmol/l, 3.210 mmol/l, 2.075 mmol/l and 0.135 mmol/l,

respectively, with no significant difference when compared to

that of controls.

3.3. Organic anions

The median and quartile range of organic anions are shown in

Table 3. The median concentrations of thiocyanate, lactate,and acetate in sialolithiasis patients were 0.220 mmol/l,

0.031 mmol/l and 0.149 mmol/l, respectively, showing no

significant difference with that in control group. However,

the level of citrate in the saliva of sialolithiasis patients was

0.007 mmol/l, significantly reduced compared to the value in

controls (P= 0.018).

3.4. Saliva flow rate and the output of each saliva ion per

minute

The submandibular/sublingual saliva flow rates of sialolithia-

sis patients and healthy controls were 0.18 (0.170.20) ml/min

and 0.29 (0.250.38) ml/min, respectively, with significantdifference (P= 0.004). The results of output of each ion per

minute showed that the outputs of potassium, magnesium,

chloride, phosphate, thiocyanate and citrate were signifi-

cantly reduced in sialolithiasis patients (P= 0.013, 0.005, 0.034,

0.041, 0.041, 0.001, respectively, Table 4).

4. Discussion

Our study revealed for the first time, to the best of our

knowledge, that the concentration of calcium ion, as a

crystallisation substance, in submandibular/sublingual saliva

was higher in submandibular sialolithiasis patients than inhealthy controls, whereas levels of magnesium and citrate

ions, as crystallisation inhibitors, were lower. Thus, increased

calcium ion and decreased citrate and magnesium ions might

be involved in the etiopathology of calculi formation.

The development of sialolithiasis is multifactorial.18 It is

believed that saliva retention due to morphoanatomic factors

Table 1 Ion chromatographic results of inorganic cations in saliva.

Inorganic cations Sialolithiasis patients Healthy controls Pvalue

Sodium (mmol/l) 4.335 (3.13013.625) 3.540 (1.58515.725) 0.880Potassium (mmol/l) 15.750 (9.52019.125) 16.150 (11.01821.400) 0.791

Magnesium (mmol/l) 0.135 (0.0990.300) 0.345 (0.1850.558) 0.044

Calcium (mmol/l) 1.035 (0.7581.558) 0.630 (0.5880.753) 0.003

Data are expressed as median and quartile range (Q25Q75).

Table 2 Ion chromatographic results of inorganic anions in saliva.

Inorganic anions Sialolithiasis patients Healthy controls Pvalue

Chloride (mmol/l) 9.095 (4.92313.175) 9.460 (5.90518.150) 0.473

Nitrate (mmol/l) 2.075 (0.0456.300) 1.645 (0.42310.553) 0.940

Phosphate (mmol/l) 3.210 (1.3634.722) 3.235 (2.0854.058) 0.821

Sulphate (mmol/l) 0.135 (0.1000.223) 0.175 (0.0730.215) 0.850


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and a high calcium concentration may lead to susceptibility of

stone formation.19 The existence of bacterial infection and

sialoadenitis can favour the nucleation and retention of

hydroxyapatitie.19 However, to date none of them has been

confirmed to play a causative role in the formation of

sialoliths, and its etiopathogenesis remains an enigma.

Electrolyte changes of saliva in sialolithiasis patients may

offer a valuable complement to the etiopathogenesis study

andcontribute to the understanding of crystallisation process.Therefore, we tried to determine whether submandibular/

sublingual saliva in submandibular sialolithiasis patients

presents major biochemical alterations compared with saliva

from healthy controls, which may offer some clues in the

pathogenesis of calculi formation.

Whole saliva collection is relatively easy to perform.

However, gingival liquid and the exudation of serum from

injured oral mucosa tissues also contribute to the electrolyte

concentrations in whole saliva.20 Therefore, we used the

glandular saliva because it is more relevant to physiology and

pathology of salivary gland than whole saliva. Among several

techniques for saliva electrolytes analysis, ion chromatogra-

phy, as a versatile separation technique, has its advantages ofsimplicity and specificity in the body fluids analysis.21,22

Previous studies demonstrated that with satisfactory repro-

ducibility and reliability, ion chromatography is well-suited to

the analysis of electrolytes in human saliva.15,16 An advantage

of analysing multiple ions in the one sample by ion

chromatography is that the interrelationships of the various

electrolytes may be examined.16 Furthermore, only a small

volumeis needed foranalysing, which is especiallysuitablefor

glandular saliva.

Our results showed that the concentrations of calcium,

citrate, and magnesium in submandibular/sublingual saliva of

patients with submandibular sialolithiasis were altered with

respect to that of normal saliva, which might be related to the

etiopathogenesis of the disease. The outputs of potassium,

magnesium, chloride, phosphate, citrate and thiocyanate

were reduced in sialolithiasis patients. However the concen-

trations of potassium, chloride, phosphate and thiocyanate

showed no changes so that their decreased outputs were due

to the lower amount of saliva. With respect to crystallisation

equilibrium, it is the ion concentration but not the output

which contributes to the crystallisation process. It has been

known that, whereas saliva is supersaturated with highconcentrations of calcium and phosphate, the precipitation

is inhibited by crystallisation inhibitors. The calcium phos-

phate crystallisation in saliva must be favoured by an

appropriate thermodynamic factor and a kinetic factor.19 An

increased level of calcium ion is the thermodynamic factor,

which provides the crystallisation driving force. And a

decreased concentration of crystallisation inhibitors serves

as a kinetic factor.19 Therefore, if the equilibrium in saliva

electrolytes is broken either by an increase in the levels of

crystallisation substances or by a decrease of crystallisation

inhibitors, precipitation of calcium phosphate will occur,

which will lead to the formation and growth of calculi. This

interprets what we found in the presentstudy. With respecttocrystallisation driving force, we found that calcium ion

showed higher concentration in submandibular/sublingual

saliva of submandibular sialolithiasis patients than in healthy

controls. At the same time, as crystallisation inhibitors,

magnesium and citrate ions exhibited lower concentrations

when compared to that of controls.

Citrate, magnesium and pyrophosphate make up most of

the inhibitors for the calcium phosphate crystal system.23

Previously, Grases et al.19 reported that calcium ion in the

whole saliva of patients with hydroxyapatite calculi was

significantlyhigherthan that found in the salivaof the healthy

group. And the salivary phytate and magnesium concentra-

tions of patients with hydroxyapatite calculi were significantly

Table 3 Ion chromatographic results of organic anions in saliva.

Organic anions Sialolithiasis patients Healthy controls Pvalue

Thiocyanate (mmol/l) 0.220 (0.1730.598) 0.340 (0.1701.145) 0.520

Citrate (mmol/l) 0.007 (0.0000.016) 0.015 (0.0100.024) 0.018

Lactate (mmol/l) 0.031 (0.0050.157) 0.170 (0.0170.243) 0.212

Acetate (mmol/l) 0.149 (0.0050.945) 0.059 (0.0080.498) 0.791


Table 4 Output of ions in saliva per minute.

Ions Sialolithiasis patients Healthy controls Pvalue

Sodium (mmol/min) 0.918 (0.761.925) 1.177 (0.5593.809) 0.579

Potassium (mmol/min) 2.795 (1.8903.341) 4.958 (3.5537.045) 0.013

Magnesium (mmol/min) 0.031 (0.0210.0476) 0.091 (0.0540.157) 0.005

Calcium (mmol/min) 0.177 (0.1330.277) 0.171 (0.1500.238) 0.821

Chloride (mmol/min) 1.826 (0.8892.555) 3.743 (1.9144.436) 0.034

Nitrate (mmol/min) 0.459 (0.1510.879) 0.634 (0.2732.143) 0.450

Phosphate (mmol/min) 0.653 (0.3700.787) 0.886 (0.6981.135) 0.041

Sulphate (mmol/min) 0.028 (0.0220.035) 0.042 (0.0250.065) 0.212

Thiocyanate (mmol/min) 0.047 (0.0340.074) 0.115 (0.0680.191) 0.041

Citrate (mmol/min) 0.001 (0.0000.002) 0.004 (0.0040.005) 0.001

Lactate (mmol/min) 0.005 (0.0020.020) 0.042 (0.0160.064) 0.096

Acetate (mmol/min) 0.037 (0.0010.078) 0.015 (0.0040.1390) 0.677



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inferior to that found in healthy group. Howeverno significant

difference of citrate concentration in whole saliva was found.

Our results of calcium and magnesium concentrations in

glandular saliva are in line with their findings in whole saliva.

Interestingly, it is well known that renal lithiasis is also

defined as the consequence of an alteration of the normal

crystallisation conditions of urine in the urinary tract.24 High

levels of calcium, oxalate, uric acid and low levels of citrate,magnesium, are considered to be the pathogenic factors in

lithiasis patients.24 Consequently, specific dietary considera-

tions have been suggested as a necessary complementary

treatmentin the clinicalmanagementof renal lithiasis patients.

Clinical studies also confirmed that oral potassium citrate or

potassiummagnesium citrate was effective in the prevention

of kidney stone formation or stone recurrence and regrowth

after extracorporeal shockwave lithotripsy and percutaneous

nephrolithotomy.2527 So, if our conclusion about electrolyte

alteration in sialolithiasis patients can be confirmed in the

future large-scale studies, oral prescription of magnesium and

citrate supplements may be considered as a simple and

convenient medical management for preventing recurrenceafter removal of sialoliths by minimally invasive surgery such

as sialendoscopy28 or extracorporeal shockwave lithotripsy.2

Themainlimitation of the presentstudy is thesmall size of

the population studied. Therefore, further work is required to

confirm these results in a large-scale prospective cohort study

as well as to clarify the exact mechanism.

In conclusion, the most novel and intriguing aspect of our

study is that the salivaelectrolyte composition of sialolithiasis

patients is substantially altered with respect to crystallisation

mechanisms. Increased calcium ion and decreased citrate and

magnesium ions may change the crystallisation equilibrium

and contribute to the crystal nucleation and growth.

Acknowledgements

Special thanks go to Dr. Z.F. Chen and Ms. S.Q. Feng (Sun Yat-

sen University, Guangzhou, China) for their technical assis-

tance and great help in ion chromatography. The authors also

thank all of the patients and the healthy volunteers who were

involved in this study.

Funding: None.

Conflict of interest: None declared.

Ethical approval: Institutional Ethic Board, Guanghua School

of Stomatology, Sun Yat-sen University.

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