Journal

of
Dentistry
Journal of Dentistry 27 (1999) 115–118

Salivary sialic acid, protein, salivary flow rate, pH, buffering capacity and
caries indices in subjects with Down’s syndrome
Ayşen Yarat a,*, Serap Akyüz b, Leyla Koç a, Hülya Erdem a, Nesrin Emekli a
a
Department of Biochemistry, Marmara University, Faculty of Dentistry, Guzelbahçe, Büyüçiftlik sok. No. 6, Nişantaşi 80200, Istanbul, Turkey
b
Department of Pedodontics, Marmara University, Faculty of Dentistry, Guzelbahçe, Büyüçiftlik sok. No. 6, Nişantaşi 80200, Istanbul, Turkey
Received 2 April 1997; accepted 30 April 1998

Abstract

Objectives: The aim of this study was to compare salivary sialic acid, protein, salivary flow rate, pH and buffering capacity and caries
indices between subjects with Down’s Syndrome and healthy controls.
Methods: Unstimulated mixed saliva was collected from 26 Down’s syndrome subjects and 25 healthy subjects of age range 6–24 years.
Total protein was determined by the method of Lowry and total sialic acid using Ehrlich reagent. Laemmli SDS–polyacrylamide gel
electrophoresis was also carried out.
Results: Buffering capacity and pH were quite similar for both groups. For permanent dentition subjects pH was significantly higher (P ¼
0.03) in the Down’s syndrome group. The salivary flow rate of the Down’s syndrome subjects was significantly lower (P ⬍ 0.01) than that of
healthy controls and the Down’s syndrome subjects’ salivary protein and sialic acid levels were significantly higher (P ⬍ 0.001). The ratios
of total sialic acid to total protein were significantly higher (P ⬍ 0.001) in the Down’s syndrome group. However, salivary sialic acid
expectoration rates, a means of compensating for flow rate differences, were significantly lower (P ¼ 0.01) in the Down’s syndrome subjects
than in controls. Electrophoresis revealed no significant differences between the protein bands of the groups. There were no significant
differences in caries indices between groups, even when compensated for age, nor in the salivary parameters within groups between sexes.
Conclusions: Total salivary sialic acid in Down’s syndrome subjects, higher in terms of levels but lower in terms of expectoration rates,
was significantly different from that of controls of similar caries indices. 䉷 1999 Elsevier Science Ltd. All rights reserved.

Keywords: Down’s syndrome; Saliva; Sialic acid; Protein; pH; Flow rate; Buffering capacity; Caries; Protein electrophoresis

1. Introduction Most of the salivary proteins are glycoproteins. The

There have been many studies on dental caries and on
periodontal diseases in Down’s syndrome (DS) subjects
[1–9], but of the salivary parameters, these studies have
generally only investigated pH and flow rate [4,5,10,11].
Studies on salivary composition of subjects with DS are
limited [10–12].
Saliva composition has been reported to be affected by
many factors: flow rate, stimulation, age, nutrition, disease,
drugs and hormones [13–18]. The major factor, however, is
hydration. If body fluid decreases by 8%, the saliva flow rate
decreases to nil. Saliva flow rate also changes according to
the physical position of subjects and has a circadian rhythm
[15,18]. One of the means by which saliva protects the teeth
against caries is by neutralizing and buffering acids. The pH
of normal saliva differs greatly between individuals [15,18].
* Corresponding author. Tel.: +90-212-233-6627/231-9120131; fax: +90-
212-246-5247.
0300-5712/99/$ - see front matter 䉷 1999 Elsevier Science Ltd. All rights reserved.
PII: S0 30 0 -5 7 12 ( 98 ) 00 0 30 - X
carbohydrate side-chains of these glycoproteins increase
resistance to proteolytic degradation. Sialic acid, which is
synthesized in the liver, is found at the end of these side-
chains. It is a major component of cell membranes
existing in conjugate form on the external surface of
the membrane. It is a component of the receptors of
insulin, interferon and serotonin and of various blood
hormones, transport proteins, lipoproteins and mucopoly-
saccharides. It is also found in various exudates [19–
21]. Sialic acid is known to affect amino acid transport on
the cell surface and to retard synaptic transmission. It plays
a role in action of various enzymes and in binding toxins
[20,21].
Elevated saliva sialic acid levels have been reported in
diabetes and cancer cases [17,22,23]. There have been, how-
ever, no studies on salivary sialic acid in DS subjects. The
aim of this study was to compare salivary total sialic acid,
protein, flow rate, pH and buffering capacity, and caries
indices between DS subjects and healthy controls.
116 A. Yarat et al. / Journal of Dentistry 27 (1999) 115–118
2. Materials and methods
This study was performed on 26 Down’s syndrome sub-
jects (11 female and 15 male, age range 7–22 years) and 25
healthy controls i.e. having no systemic diseases (11 female
and 14 male, age range 6–24 years). The DS subjects were
randomly selected from private or state schools for
handicapped children in Istanbul, Turkey.
Dental examinations were conducted by the dentist under
natural lighting with the aid of a dental mirror and explorer
and dmf-t, DMF-T, dmf-s, DMF-S indices were assessed
according to the WHO criteria [24]. Unstimulated mixed
saliva samples were collected by expectoration into a
funnel, after an overnight fast and after the mouth had
been rinsed with distilled water.
The saliva samples were collected over a 5-min period
and the average saliva flow rate calculated from the total
volume. The saliva samples were analyzed for pH and buf-
fering capacity [18] by using pH paper. To determine pro-
tein and sialic acid, saliva samples were centrifuged at 2500
⫻ g for 10 min at room temperature. The supernatants were
kept at ¹ 20⬚C. Salivary protein was determined by the
method of Lowry [25].
Salivary total sialic acid was determined using Ehrlich
reagent [21,26]. 0.2 ml supernatant and 5% HClO 4
(1.5 ml) were placed in a tube and mixed using a
vortex mixer. This was then incubated in boiling water
for 5 min and 1 ml Ehrlich reagent (0.7 g p-dimethylamino-
benzaldhyde þ250 ml 6 M HCl) added into it. After being
kept in a boiling water bath for 15 min it was cooled and
1 ml of distilled water was added. The absorbances of the
saliva samples and a standard (sialic acid, Fluka Switzer-
land, code: 132697b-k A5011 39) at 525 nm were recorded
and the total sialic acid content of saliva calculated.
Protein electrophoresis was carried out as described by
Laemmli [27]. Schleicher and Schuller profile system mini
electrophoresis was performed using a Sigma low molecular
weight protein standard (SDS-7, Dalton Mark VII-L). Saliva
samples were prepared for SDS polyacrylamide slab gels.
They were diluted with sample buffer and then heated at
Table 1
Mean levels of salivary parameters in Down’s syndrome and healthy control groups
Control group
(n ¼ 25)
Mean SD
Age (year) 12.44 5.49
Salivary flow rate 0.67 0.23
(ml min ¹1)
Salivary pH 7.3 0.41
Salivary buffering 3.82 0.66
capacity
Protein (g l ¹1) 1.54 0.66
Sialic acid (g l ¹1) 0.10 0.06
SD, standard deviation.
90–100⬚C for 5 min. From each sample 10–30-ml portions
containing 20 mg proteins were placed into the slab gel well.
After electrophoresis, scans of Coomassie Brillant Blue
(0.1% Coomassie Brillant Blue R-250, 40% methanol,
10% acetic acid, Sigma, St Louis, MO, USA) stained
protein bands from slab gels obtained by a densitometer
(Helena laboratories TCL-plus) were measured with a
planimeter (Placom-Sokkisha, Kp-90N, digital) and the
percentages of each protein band were calculated.
Differences between groups were compared using
unpaired t-tests and correlation analysis was performed on
all parameters.
3. Results
The mean levels of all parameters measured are given in
Table 1. This shows that there were no significant differ-
ences in mean age, salivary pH and buffering capacity
between DS subjects and controls but that flow rate in the
DS group was significantly lower (P ⬍ 0.001) and protein
and sialic acid levels were significantly higher in the DS
group (P ⬍ 0.001) (Table 1). Similar differences between
these groups were found when dividing each group into
mixed dentition (age ⬍ 13) or permanent dentition (age
ⱖ 13) groups. There were, however, some differences in
the permanent dentition subjects. Salivary pH in the DS
group was significantly higher than controls (P ¼ 0.03)
and protein, although still higher in the DS group, failed
to reach statistical significance (P ¼ 0.07) (Table 2). The
Laemmli SDS PAGE protein bands, which were very simi-
lar to those described in our previous studies [13,28], did not
differ significantly between groups (Fig. 1). Caries indices
were not significantly different between groups (P ⬎ 0.5). In
order to compensate for age dependency, each index was
divided by age (in years). These did not differ between
groups either (P ⬎ 0.4). When comparing between sex
within groups, there were no significant differences in the
salivary parameters.
Correlations between sialic acid, protein and salivary
Down’s syndrome
group (n ¼ 26)
Mean SD P
12.31 3.94 0.92
0.11 0.13 ⬍ 0.001
7.4 0.57 0.55
3.63 0.69 0.33
3.26 2.07 ⬍ 0.001
0.29 0.20 ⬍ 0.001
 A. Yarat et al. / Journal of Dentistry 27 (1999) 115–118 117

Table 2
Salivary parameters according to dentition

Control group Down’s syndrome

(n ¼ 16) group (n ¼ 17)

Mean SD Mean SD P

Mixed dentition (age ⬍ 13)

Salivary flow rate 0.75 0.22 0.11 0.14 ⬍ 0.001
(ml min ¹1)
Salivary pH 7.41 0.42 7.32 0.66 0.67
Salivary buffering 4.00 0.41 3.67 0.72 0.11
capacity
Protein (g l ¹1) 1.32 0.32 3.50 2.46 ⬍ 0.001
Sialic acid (g l ¹1) 0.11 0.06 0.31 0.24 ⬍ 0.001
(n ¼ 9) (n ¼ 9)
Permanent dentition (age ⱖ 13)
Salivary flow rate 0.54 0.19 0.09 0.12 ⬍ 0.001
(ml min ¹1)
Salivary pH 7.11 0.33 7.5 0.35 0.03
Salivary buffering 3.5 0.90 3.72 0.57 0.54
capacity
Protein (g l ¹1) 1.92 0.93 2.78 0.94 0.07
Sialic acid (g l ¹1) 0.08 0.06 0.26 0.09 ⬍ 0.001

flow rate are given in Table 3. The correlation between
salivary sialic acid and protein levels was good for DS sub-
jects and all subjects but poor for controls. Measurement of
total sialic acid of course involved release of bound sialic
acid. In order to take this into account, ratios of total sialic
acid to total protein were calculated. The average ratio for
the DS group was 0.10 and 0.07 for controls. The differ-
ences in these ratios were still very significant (P ⬍ 0.001)
and may indicate a difference in free sialic acid.
Medium negative correlations were found between
salivary flow rate and protein levels (r ¼ ¹0.53) and
between salivary flow rate and sialic acid levels (r ¼
¹0.51) for all subjects. The correlation between
salivary flow rate and protein levels was good (r ¼
¹0.68) for controls but very poor (r ¼ ¹0.15) for DS
subjects. The correlation between salivary flow rate and
Fig. 1. Typical SDS polyacrylamide gel electrophoretic patterns of salivary
proteins (s, Standard protein mixture; DS, Down’s syndrome group; C,
healthy control group).
sialic acid levels was poor for controls and nil for the DS
group (r ¼ ¹0.01).
Multiplying total sialic acid levels by salivary flow rate
for each subject, i.e. calculating the sialic acid expectoration
rate, was used to take salivary flow rate differences into
account. Average rates were 0.03 mg min ¹1 for the DS
group and 0.07 mg min ¹1) for controls. The differences in
these rates between groups were significant (P ¼ 0.01).
4. Discussion
As the composition and physiology of saliva quite clearly
influences oral health and often indicates systemic
conditions, it warrants thorough investigation.
Some studies have found differences in salivary pH and
flow rate between control and DS subjects [5,11], others
have not [4,10,11]. This study found no significant
differences in pH between groups but found salivary flow
rate to be significantly lower in the DS group. However, in
the permanent dentition subjects, salivary pH in the DS
group was significantly higher than in controls (P ¼ 0.03).
There were no significant differences in buffering capacity
between groups.
Some studies have reported dental caries in Down’s syn-
drome subjects as being lower [5–7], higher [8] than or not
different [9] to that in healthy controls. The present study
found no significant differences in caries indices between
groups.
This study found significantly elevated salivary sialic
acid in Down’s syndrome subjects and although protein
was elevated in those with a permanent dentition, this did
not reach statistical significance. Akyüz et al. [28] found
elevated salivary protein levels in diabetic children and
118 A. Yarat et al. / Journal of Dentistry 27 (1999) 115–118

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