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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:drliaoguiqing@hotmail.com(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:drliaoguiqing@hotmail.comhttp://dx.doi.org/10.1016/j.archoralbio.2009.11.006http://dx.doi.org/10.1016/j.archoralbio.2009.11.006mailto:drliaoguiqing@hotmail.com8/14/2019 1-s2.0-S0003996909002957-main.pdf
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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
Data are expressed as median and quartile range (Q25Q75).
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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
Data are expressed as median and quartile range (Q25Q75).
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
Data are expressed as median and quartile range (Q25Q75).
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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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