2013 John Wiley & Sons A/S.

Published by John Wiley & Sons Ltd

J Periodont Res 2014; 49: 129136

All rights reserved

JOURNAL OF PERIODONTAL RESEARCH

doi:10.1111/jre.12088

Salivary antioxidants as
periodontal biomarkers in
evaluation of tissue status
and treatment outcome

N. Novakovic1, T. Todorovic2,
M. Rakic1, I. Milinkovic1,
I. Dozic2, S. Jankovic1,
Z. Aleksic1, S. Cakic1
1

Department for Periodontology and Oral

Medicine, Faculty of Dental Medicine,
University of Belgrade, Belgrade, Serbia and
2
Department of Biochemistry, Faculty of Dental
Medicine, University of Belgrade, Belgrade,
Serbia

Novakovic N, Todorovic T, Rakic M, Milinkovic I, Dozic I, Jankovic S, Aleksic

Z, Cakic S. Salivary antioxidants as periodontal biomarkers in evaluation of
tissue status and treatment outcome. J Periodont Res 2014; 49: 129136. 2013
John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Background and objective: One of the major pathologic patterns in periodontitis
represents an imbalance among the production of free radicals and local antioxidants resulting in periodontal tissue destruction. The objective of the study was
to investigate the inuence of non-surgical periodontal treatment on salivary
antioxidants and to evaluate their capacity as biomarkers reecting periodontal
tissue condition and therapy outcome.
Material and Methods: Sixty-three systemically healthy non-smokers, including
21 periodontally healthy subjects (HC) and 42 patients with current chronic
periodontitis fullled the inclusion criteria. Half of the patients received scaling
and root planing (SRP) and the other half received only oral hygiene instructions. Full mouth clinical measurements, including gingival index (GI), plaque
index (PI), periodontal pocket depth, clinical attachment level and saliva sampling were performed at baseline visit and 2 mo after treatment/baseline visit.
Total antioxidant capacity (TAOC), albumins (ALB), uric acid (UA), superoxide dismutase (SOD) and glutathione peroxidase (GPX) were evaluated in saliva
samples using commercial kits.
Results: All measured antioxidants were aected by treatment resulting in significant increase in TAOC (p < 0.005), ALB (p < 0.001), UA (p < 0.001) and GPX
(p < 0.001) and decrease of SOD (p < 0.005) in response to SRP, where no differences were observed for any of parameters in the oral hygiene instructions
group. Comparison of antioxidant levels between the HC and SRP group
showed that before treatment ALB were signicantly higher in HC when compared to the SRP group (p = 0.039), and GXP (p = 0.000) and SOD (p = 0.021)
levels were signicantly higher in the SRP group. Comparison of values after
treatment showed that TAOC was signicantly higher in the HC than in the
SRP group (p = 0.001), but UA was, inversely, signicantly higher in the SRP
group (p = 0.034). All clinical parameters except clinical attachment level were
signicantly decreased after SRP and signicant correlations were observed
between SOD and GI (p = 0.017), SOD and PI (p = 0.011), GPX and GI
(p = 0.003) and GPX and PI (p = 0.008).

Mia Rakic, DDS, PhD, Department for

Periodontology and Oral Medicine, Faculty of
Dental Medicine, University of Belgrade,
Dr Subotica 4 Street, 11000 Belgrade, Serbia
Tel: +381 11 3629201
Fax: +381 11 3065463
e-mail: mia.rakic@gmail.com
Key words: antioxidants; biomarkers;

periodontitis; total antioxidant status; albumin;

superoxide dismutase; glutathione peroxidase;
uric acid
Accepted for publication March 21, 2013

130

Novakovic et al.

Conclusion: Non-surgical periodontal treatment aected salivary TAOC, ALB,

UA, SOD and GPX; moreover, these biochemical parameters convincingly
reected periodontal status and tissue response on treatment.

Periodontitis is a chronic inammatory

disease characterized by excessive host
response on periodontal pathogens
resulting in periodontal tissue destruction (14). Reactive free radicals being
the main tool of the host response in
combating infection play an important
role in eliminating periopathogens (5).
Polymorphonuclear cells and monocytes, as well as other inammatory
cells such as endothelial cells, broblasts and osteoclasts produce free
radicals on bacterial challenge (69),
therewith resulting in respiratory
burst, which is the central pathway of
phagocytosis (10). Regarding periodontal inammation characterized
with extreme concentrations of proinammatory mediators, the inuence of
periodontal inammation on phagocytes was investigated and showed that
the proinammatory cytokines directly
activate the respiratory burst in neutrophils (11) indicating that they are
an enhancing factor in local oxidative
stress in periodontitis. Finally, tissue
destruction occurs because of imbalance between the released free radicals
and antioxidants (12) where unneutralized free radicals react with tissue
aecting its structure, function and
biological behavior. Previous studies
on antioxidants in patients with periodontitis have already reported
decreased antioxidant levels in that
population when compared to healthy
subjects in both gingival crevicular
uid and saliva (13,14).
The main biologic concept in periodontal treatment is oriented towards
reduction of causative agents and
their consequences with the main aim
being to stimulate and support the
regenerative potential of periodontal
tissues due to inammation reduction.
When considering oxidative stress, the
main trend in periodontal therapy is
the reduction of free radicals and
increase of antioxidants during their
neutralization and re-obtaining the
oxidative equilibrium in periodontal

tissues. Based on structural and functional characteristics, there are two

types of salivary antioxidants, including enzymatic and non-enzymatic
antioxidants, where the enzymatic are
responsible for direct neutralization of
free radicals and the non-enzy
matic neutralize secondary oxidative
products.
Recent periodontal research is concerned with the determination of biomarkers that accurately reect tissue
status during diagnostics and evaluation of treatment outcome. Related to
that, gingival crevicular uid has been
accepted as the diagnostic medium of
choice, which convincingly reects
periodontal tissue condition (15,16).
On the other hand, this method is a
highly sensitive but expensive method
requiring special equipment and professionally skilled persons, where
every step aects the outcome, making it complicated for routine clinical
practice. Of these reasons, saliva was
considered as an experimental medium for evaluation of biomarkers.
Saliva sampling represents an easy,
non-expensive protocol causing minimal discomfort of the subject (17).
Thus, the objective of this study was
to investigate the inuence of nonsurgical periodontal treatment on salivary antioxidants and to assess their
diagnostic use in monitoring periodontal
tissues
and
treatment
outcome.

Material and methods

This was a randomized controlled
study that included patients visiting
the Clinic for Periodontology and
Oral Medicine of the Faculty of Dental Medicine, University of Belgrade
between September 2010 and December 2011. The protocol was approved
by the Ethics Committee of the Faculty of Dental Medicine, University
of Belgrade (permission reference no.
36/47).

Patient selection

The study population included systemically healthy non-smokers older

than 25 years, among them were
periodontally healthy subjects and
patients with untreated moderate
chronic periodontitis (18) with radiographic evidence of generalized alveolar bone loss > 30%, presence of at
least one pocket with periodontal
pocket depth (PPD) > 5 mm per
quadrant with positive bleeding on
probing and presence of at least three
teeth per quadrant were included.
Exclusion criteria were: (i) received
periodontal treatment in the last
1 year; (ii) antibiotic intake in previous 3 mo; (iii) use of non-steroidal
drugs in previous 1 mo; (iv) pregnant
or lactating women; and (v) use of
vitamins C and E in previous 3 mo.
Study design

In the screening visit all participants

were periodontally examined to determine if they were fullling the inclusion criteria, they were informed on
study procedures and gave their written consent. After that, patients were
randomly assigned using a computergenerated table to receive oral hygiene
instructions (OHI) or scaling and root
planing (SRP), and they were scheduled for baseline visit in the next 48 h.
At the baseline visit, the rst saliva
sample was collected (see Saliva collection) before clinical measurements to
avoid any potential contamination of
specimen by blood originating from
periodontal pockets after provocation
with clinical measurements. After sampling, full-mouth clinical measurements (see Clinical measurements)
were performed and recorded into
patients study chart. Depending
of group aliation, patients were
instructed on oral hygiene or they
underwent non-surgical periodontal
treatment. OHIs included standardized

Salivary biomarkers related to periodontitis

instructions: use of manual toothbrush with demonstrated technique of
tooth brushing and appropriate interdental brushes (Curaprox interdental
brushes; Curaprox, Curaden International AG, Kriens, Switzerland).
Non-surgical periodontal treatment
included OHIs and full-mouth SRP.
Treatment and clinical measurements
were performed by the same periodontist (N.N.) under local anesthesia aiming to avoid a painful experience
during periodontal debridement that
included SRP using ultrasonic scaler
and Gracey curettes. Whole treatment
was completed in two visits within 7 d.
All patients in the control group
received non-surgical periodontal
treatment after the study period.
The follow-up visit was scheduled
2 mo after treatment/baseline visit for
the OHI group. The second saliva
sample was collected and the same
clinical measurements were performed
and recorded in the chart. Patients in
the SRP group were reinstructed on
oral hygiene and patients in the control group underwent non-surgical
periodontal treatment.
Clinical measurements

Clinical examination was performed by

one single-blinded calibrated examiner
(N.N.) blinded to the participant group
assignments. Intra-examiner calibration was performed before and during
the study, by assessing PPD and clinical attachment level (CAL), with a
degree of agreement within  1 mm
higher than 85% for both tests. The
following clinical parameters were
assessed using a graduated periodontal
probe (North CarolinaHu-Friedy,
Chicago, IL, USA) in six points: gingival index (GI) (19), and plaque index
(PI) (20), pocket depth distance from
the gingival margin to the location of
the tip of the periodontal probe
inserted in the pocket with moderate
probing force and CAL as the distance
from the cemento-enamel junction to
the location of the inserted probe tip.
Saliva collection

The samples for biochemical analyses

were collected at baseline visit and

2 mo after nal periodontal treatment/

baseline visit for the OHI group.
Unstimulated whole expectorated saliva (5 mL) was collected from each
subject between 09:00 and 10:00 h,
where the patients were ordered not to
eat anything before sampling. Subjects
rinsed their mouth with water before
sampling, then they expectorated
saliva into sterile tubes (Salivette,
Sarstedt, Germany) specialized for this
kind of sampling while seated in an
upright position. Collected samples
were immediately transported to the
laboratory where they were centrifuged at 4000 g for 10 min and frozen
at 80C until analyzing.
Determination of antioxidants in
saliva samples

Antioxidants were mostly determined

using the colorimetric method and
commercial kits for the determination
of: superoxide dismutase (SOD;
Ransod; Randox Laboratories Ltd.,
Crumlin, UK), albumins (ALB,
Albumin liquicolor; Human Gesellschaft f
ur Biochemica und Diagnostica mbH, Wiesbaden, Germany), uric
acid (UA, uric acid liquicolor; Human
Gesellschaft f
ur Biochemica und
Diagnostica mbH), total antioxidant
capacity (TAOC, total antioxidant
status; Randox Laboratories Ltd.)
and GPX activity (Ransel; Randox
Laboratories Ltd.) was particularly
determined using the ultraviolet
method.
Statistical analysis

Statistical analysis was performed

using commercial statistical software
(SPSS 18.0, Inc., Chicago, IL, USA).
Primary outcome variables were PPD
changes and changes in measured
antioxidant parameters (ALB, UA,
GPX, SOD and TAOC). Biomarker
levels were expressed as follows: ALB
as g/L, UA and TAOC as lM, and
GPX and SOD as IU/L. Secondary
outcome variables included CAL, GI
and PI. Clinical variables (GI, PI,
PPD and CAL) were calculated by
patient and visit and then by group.
The PPD variable was also divided in
to two categories (13 and 46 mm)

131

and proportions for both were recalculated at each follow-up point. All
data were expressed as mean and SD.
During study planning, the sample
size calculation was performed for
PPD considering a SD of 0.8 mm and
a desirable dierence between groups
of 1.02 mm, with 90% of power,
resulting in a sample of 14 patients
per arm (21).
The chi-squared test was performed
for all continuous variables to assess
normality of the data. After that,
parameters between groups were analyzed using the t-test. In assessment of
dierences in measured parameters
between baseline and follow-up visit,
the paired t-test was used for each
group separately. In the assessment of
correlation between parameters, Pearsons correlation coecient was used.
Demographic data (gender and age) as
nominal variables were analyzed using
the chi-squared test. Comparison of
UA distribution among sexes was
evaluated using the MannWhitney
test. Statistical signicance was established at the 95% condence level.

Results
Patients characteristics

The study population included 63

subjects, i.e. 21 healthy subjects and
42 patients with chronic periodontitis;
between them 21 received non-surgical
periodontal treatment (aged 39.2 
11.5) and 21 OHI (aged 38.1  10).
Demographic, clinical and biochemical parameters were similar and without statistical signicance at baseline
between groups (Table 1).
Values of periodontal and
antioxidant parameters at follow-up
points

Table 2 depicts dierences in measured clinical and biochemical parameters among baseline and second
follow-up points of the treated and
control group. All measured clinical
parameters except CAL showed a statistically signicant reduction after
periodontal treatment in the SRP
group, contrary to that, in the OHI
group clinical parameters remained

132

Novakovic et al.

Table 1. Demographic and clinical characteristics of the groups

HC

OHI

SRP

p-Value

Age (years)

35.2 (7.1)

38.8 (12.4)

39.2 (11.5)

OHI/SRP = 0.264
OHI/HC = 0.384
SRP/HC = 0.386

Gender (%)
Male
Female
PI

14 (66.67)
7 (33.33)
0.33  0.27

14 (66.67)
7 (33.33)
1.41  0.52

14 (66.67)
7 (33.33)
1.38  0.55

GI

0.24  0.21

1.83  0.10

1.96  0.41

PPD

2.11  1.67

3.37  0.51

3.39  0.66

PPD 13 mm (%)
PPD 46 mm (%)
CAL

0.00

61.4  10.3
37.3  14.7
3.00  1.00

62.3  10.8
36.2  9.8
3.00  1.00

p = 1.00
OHI/SRP
OHI/HC
SRP/HC
OHI/SRP
OHI/HC
SRP/HC
OHI/SRP
OHI/HC
SRP/HC
OHI/SRP
OHI/SRP
OHI/SRP
OHI/HC
SRP/HC

=
=
=
=
=
=
=
=
=
=
=
=
=
=

0.729
0.023*
0.019*
0.112
0.012*
0.019*
0.543
0.047*
0.045*
0.268
0.257
0.485
0.000*
0.000*

CAL, clinical attachment level; GI, gingival index; HC, healthy controls; OHI, group
receiving oral hygiene instructions; PI, plaque index; PPD, periodontal pocket depth; SRP,
group receiving scaling and root planing.
All values are expressed as means  SD, except gender distribution, which is expressed in
percentages.
*p < 0.05.

unchanged after 2 mo. Regarding

biochemical parameters, in the
SRP group UA, ALB, GPX and
TAOC were signicantly increased in
response to periodontal treatment,
and SOD was signicantly decreased.
In the OHI group, all antioxidants
remained unchanged after 2 mo.

Comparison of antioxidant levels

between the HC and SRP group are
listed in Table 3. The analysis of values before treatment showed that
ALB levels were signicantly higher
in the HC when compared to the SRP
group
(p = 0.039),
and
GXP
(p = 0.000) and SOD (p = 0.021)

levels were signicantly higher in the

SRP group. Comparison of values
after treatment showed that TAOC
was signicantly higher in the HC
than in the SRP group (p = 0.001),
but UA was, inversely, signicantly
higher in the SRP group (p = 0.034).
The analysis of distribution of UA
among women and men did not show
signicant dierence (p = 0.237).
The analysis of correlation between
measured antioxidants is outlined in
Table 4. The signicant positive correlation was observed between UA
and TAOC (p = 0.019) and signicant
negative correlations were reached
between GPX and ALB (p = 0.043)
and GPX and SOD (p = 0.035).
Correlations among salivary biomarkers and clinical parameters were
calculated for each follow-up visit. At
baseline visit positive correlations
were observed between SOD and GI
(R = 0.525, p = 0.017) and between
GPX and PI (R = 0.575, p = 0.008)
(Table 5). At post-treatment followup, no signicant correlation was
established.

Discussion
Results of this study have shown that
non-surgical periodontal treatment
aects all measured salivary antioxidants. Non-surgical treatment resulted
in an increase of TAOC, together

Table 2. Changes in measured parameters from baseline to 2 mo of follow-up in treatment and control groups
OHI (n = 21)
Baseline
PI
GI
PPD
CAL
PPD
13 mm (%)
PPD
46 mm (%)
ALB
UA
GPX
SOD
TAOC

1.4
1.8
3.37
3.00
61.4

After treatment






0.5
0.1
0.51
1.00
10.3

37.3  14.7
1.30
153.9
1842.90
0.42
0.40

SRP (n = 21)







0.12
40.91
157.42
0.37
0.35

1.3
1.8
3.37
3.00
61.4







p-Value

Baseline

After treatment






0.7
0.1
0.73
1.00
10.3

0.543
1.000
0.140
1.000
1.000

1.38
1.96
3.39
2.94
62.3

37.5  14.7

1.000

36.2  9.8







0.647
0.729
0.512
0.411
0.607

1.32
154.00
1841.82
0.37
0.52

0.31
40.73
157.27
0.31
0.22

1.31
153.95
1842.95
0.45
0.40







0.55
0.41
0.66
0.99
10.8

0.06
41.87
157.66
0.12
0.23

0.35
0.54
2.02
2.93
94.7







0.29
0.24
0.53
1.01
4.6

6.3  3.5
1.42
198.42
3310.75
0.39
0.65







0.11
87.73
169.24
0.23
0.35

p-Value
< 0.001*
< 0.001*
< 0.001*
0.185
< 0.001*
< 0.001*
<
<
<
<
<

0.001*
0.001*
0.001*
0.005*
0.005*

ALB, albumins; CAL, clinical attachment level; GI, gingival index; GPX, glutathione peroxidase; OHI, group receiving oral hygiene
instructions; PI, plaque index; PPD, periodontal pocket depth; SOD, superoxide dismutase; SRP, group receiving scaling and root planing; TAOC, total antioxidant capacity; UA, uric acid.
*Statistical signicance determined by t-test.

Salivary biomarkers related to periodontitis

133

Table 3. Comparison of antioxidant concentrations among the HC and SRP groups

HC
Baseline
After treatment
p-Value
HC/baseline
HC/after treatment

ALB

UA

GXP

SOD

TAOC

1.42  0.12
1.31  0.06
1.42  0.11
0.039*
0.242

198.43  87.73
153.95  41.87
198.42  87.73
0.707
0.034

1869,43  185.31
1842.95  157.66
3310.75  169.24
0.000*
0.203

0.37  0.14
0.45  0.12
0.39  0.23
0.021*
0.744

0.59  0.14
0.40  0.23
0.65  0.35
0.497
0.001*

ALB, albumins; GPX, glutathione peroxidase; HC, healthy controls; SOD, superoxide dismutase; TAOC, total antioxidant capacity;
UA, uric acid.
*Statistical signicance.

Table 4. Correlations among measured antioxidants

GPX

SOD

0.456, p = 0.043*
0.412, p = 0.071

0.474, p = 0.035*

ALB
UA
GPX
SOD

TAOC

0.284, p = 0.225
0.084, p = 0.724
0.474, p = 0.035*

0.155,
0.846,
0.249,
0.228,

p
p
p
p

ALB
=
=
=
=

0.515
0.001*
0.289
0.334

0.604, p = 0.005*
0.232, p = 0.325
0.279, p = 0.234

ALB, albumins; GPX, glutathione peroxidase; SOD, superoxide dismutase; TAOC, total
antioxidant capacity; UA, uric acid.
*Statistical signicance established using Pearsons correlation coecient.

Table 5. Correlation among antioxidants

and clinical parameters at baseline
PI
UA
R
0.202
p
0.383
ALB
R
0.357
p
0.112
GPX
R
0.575*
p
0.008
SOD
R
0.356
p
0.123
TAOC
R
0.171
p
0.473

GI

PPD

CAL

0.109
0.638

0.065
0.778

0.141
0.541

0.742
0.751

0.002
0.992

0.143
0.535

0.367
0.112

0.252
0.282

0.001
0.996

0.525*
0.017

0.004
0.987

0.175
0.46

0.069
0.773

0.312
0.898

0.16
0.531

ALB, albumins; GPX, glutathione peroxidase; SOD, superoxide dismutase; TAOC,

total antioxidant capacity; UA, uric acid.
*Statistical signicance established using
Pearsons correlation coecient.

with an increase of non-enzymatic

antioxidants (UA, ALB) and GPX,
but also in a signicant decrease of
SOD. This result was additionally
supported by comparison of antioxidant levels in the SRP group before
and after treatment with values in
healthy individuals, showing that relations of values among patients and

healthy individuals had completely

changed after treatment. A signicant
correlation has been achieved for
GPX and PI as well as SOD and GI.
Overall results show that only SOD
behaved in a dierent way, as it was
the only antioxidant positively
correlated with inammation. Furthermore, SOD showed a negative
correlation with GPX, which additionally supports its dierently characterized appearance when compared
to other antioxidants.
Several studies have reported a
decreased antioxidant capacity in subjects with periodontitis in both saliva
(14) and gingival crevicular uid (13).
Moreover, Guentsch et al. (14)
reported results on salivary TAOC
and GPX in response to non-surgical
periodontal treatment, where TAOC
remained unchanged while GPX was
signicantly decreased after treatment,
which is discordant with our ndings.
In a similar study, Brock et al. (13)
investigated TAOC in gingival crevicular uid and saliva of chronic periodontitis and healthy subjects and
compared the ndings. These authors
reached a signicantly lower TAOC
concentration in gingival crevicular
uid samples of patients suering
chronic periodontitis, but not in saliva

samples of the same patients, moreover the TAOC concentration was

signicantly lower in saliva samples
when compared to gingival crevicular
uid. In these studies, the authors did
not obtain a compatible correlation
for salivary and gingival crevicular
uid TAOC. However, Chapple et al.
(22) reported unchanged TAOC in
plasma in response to non-surgical
periodontal treatment but a signicant increase of TAOC in gingival
crevicular uid, which corresponds to
our ndings in saliva. When comparing our results in saliva with ndings
of aforementioned researchers in
gingival crevicular uid, we nd
that results are compatible indicating
that salivary TAOC measured using
applied protocols convincingly reects
tissue status. A possible explanation
for this inconsistency among gingival
crevicular uid and salivary levels in
cited studies could be that dierent
luminescence techniques and diagnostic kits applied in these studies were
not sensitive enough to reach changes
in saliva. This explanation is related
to saliva having a lower concentration
of mediators compared to blood
and other biouids such as gingival
crevicular uid. Simultaneously, we
obtained results compatible with those
reported for a gingival crevicular uid
medium (being the most accurate one
for this kind of analysis), which suggests that our protocol based on spectrophotometry in conjunction with
applied kits is sensitive enough to
provide accurate results on periodontal tissue status. The signicance of
this nding is that it indicates compatibility of gingival crevicular uid
and saliva ndings, and suggests
saliva is an accurate biouid for

134

Novakovic et al.

evaluation of therapy outcome of this

biomarker under the applied protocol.
However, according to these ndings
TAOC was signicantly higher in
the HC when compared to levels
after SRP treatment, which may be
explained as a consequence of posttreatment GPX and SOD decrease.
GPX was usually found to be signicantly increased in periodontitis compared to healthy individuals (23,24),
which correspond to our ndings
where GPX was signicantly higher in
SRP before treatment when compared
to HC. On the other hand, Tsai et al.
(25) reported no dierences in GPX
concentration between periodontal
inammation and healthy tissues.
Furthermore, Guentsch et al. (14)
reported a GPX decrease in response
to non-surgical periodontal treatment.
This discrepancy between previously
reported results and our ndings
might be explained by dierent determination protocols/assays applied
among the studies as well. When considering the correlation between GPX
and clinical parameters, the single signicant correlation was observed with
PI, which corresponds to ndings of
Wei et al. (26).
In regard to SOD, this antioxidant
was reported to be positively associated with periodontitis (22) based on
the fact that it was often considered
as a marker of oxidative stress. Our
ndings of SOD being signicantly
higher before SRP treatment when
compared to periodontally healthy
subjects are in accordance with Wei
et al. (26). Moreover, Wei et al. (26)
showed that SOD decreases in
response to non-surgical periodontal
treatment in both gingival crevicular
uid and saliva, which corresponds to
our ndings in saliva, and supports
saliva as an accurate diagnostic uid
for this biomarker.
Regarding non-enzymatic antioxidants as indicators of periodontal
condition, Panjamurthy et al. (23)
investigated vitamin C and vitamin E
in plasma and erythrocytes, and
reduced glutathione in the same samples as well as in gingival tissue of
periodontitis and healthy subjects.
These authors reported signicantly
lower values of all measured antioxi-

dants in all samples of patients with

periodontitis with the exception of
reduced glutathione in gingival samples, which was signicantly higher
in periodontitis when compared to
healthy individuals. Furthermore,
Chapple et al. (22) investigated the
correlation of serum levels of vitamin
A, C, E, UA, a-carotene, b-carotene,
selenium, lutein, b-cryptoxanthine and
bilirubin and reported a negative correlation between serum antioxidant
capacity and periodontal risk. In
another study, Iwasaki et al. (27)
reported that low serum levels of
ALB are associated with progression
of periodontitis, suggesting this protein as an indicator of periodontal
risk. This result is compatible with
our nding whereby ALB increased in
response to SRP therapy and
remained lower in these patients when
compared to healthy individuals
despite treatment. Simultaneously,
UA signicantly increased on SRP
treatment while after treatment it was
signicantly higher in periodontitis
when compared to periodontally
healthy subjects. Considering these
results as a whole, it may be suggested that non-enzymatic antioxidants are promising indicators of
periodontal health as ALB were signicantly higher in HC than at the
baseline in the SRP group and UA
was the single antioxidant signicantly higher after SRP when compared to HC. This is the rst study to
report a positive association of salivary ALB and UA with successful
non-surgical periodontal treatment.
The equilibrium among free radicals and antioxidants in tissues as a
necessary precondition for periodontal health is found to be disturbed in
periodontitis. This occurs as a result
of excessive amounts of free radicals,
locally produced on strong and continual periodontal inammation (28)
that exceeds and exhausts local antioxidant capacities leading to tissue
damage. Free radicals in periodontitis
originate from inammatory cells
(mostly from phagosomes) (9,2931)
as their main tool in the elimination
of pathogens, but as phagocytosis in
periodontitis is limited and frequently unsuccessful based on specic

patterns of periopathogens resistance,

it results in the accumulation of free
radicals in periodontal tissues. By taking this into consideration, it is reasonable to expect that elimination of
periodontal inammation decreases
free radicals by facilitating antioxidants to neutralize them and therewith contributing in re-obtaining
oxidative homeostasis in the tissues.
In our study, we found that both enzymatic and non-enzymatic antioxidants
have been aected by inammation
during their low tissue concentrations.
Our results proved that both enzymatic and non-enzymatic antioxidants
signicantly increased with the reduction of inammation. This nding
indicating that non-enzymatic antioxidants were exhausted due to elimination of secondary oxidative products,
suggests UA and ALB as markers of
tissue damage. The interesting observation in ALB behavior was its negative correlation with GPX (R =
0.456, p = 0.043) and with UA
(R = 0.604, p = 0.005). As ALB is
known as a major antioxidant in
plasma and a sponge of the circulation (32), the possible explanation
could be in the expressed anity to
ligands (secondary oxidative products
in this case) when compared to other
two antioxidants with a subsequently
greater consumption of ALB. The
single antioxidant that showed a
decrease during periodontal treatment
was SOD, which corresponds to its
characteristic compensatory increased
concentration under inammation
(3335), and thereby expressing its
major role of primary antioxidant
defense in tissue protection under oxidative stress. In addition to this, SOD
was signicantly higher in the SRP
group at baseline when compared to
healthy controls whereby this signicance did not persist after treatment.
On the other hand, GPX is a major
enzyme in the scavenging of H2O2
generated by SOD in cytosol and
mitochondria, which is negatively correlated to SOD in our study. Simultaneously it is recognized as an indirect
marker of oxidative stress as there is
usually a compensatory increase
together with SOD as a response to
inammatory stimulation. However,

Salivary biomarkers related to periodontitis

in our study salivary GPX was negatively correlated to SOD and signicantly decreased under inammation
indicating a dierently dened pattern
in periodontal inammation. Regarding inter-relations between TAOC
and other measured antioxidants all
measured antioxidants remained positively correlated to TAOC except
ALB, but the single statistically significant correlation
was observed
between UA and TAOC. The potential reason for establishing signicant
correlation only between UA and
TAOC could be the predominance of
male subjects in the SRP group
(67%) as UA is normally higher in
men (36). However, we have not been
able to obtain a statistical signicance
between males and females, which
may have been a consequence of the
limitation by size.
Determination of reliable periodontal molecular markers measurable
in saliva bares precious signicance
regarding numerous specic pathophysiologic conditions present in periodontal pockets that aect sensitive
methods usually used for the determination of biomarkers in gingival
crevicular uid (such as ELISA, RT
PCR or other commercial kits and
methods). Furthermore, gingival crevicular uid sampling requires specic
sensitive techniques and equipment
for quantication of biomarkers. In
the clinical sense this could present
diculty as the mandatory requirements are a complete absence of saliva and blood, which is often dicult
to achieve with active periodontal
pockets or in newly formed periodontal tissue. On the other hand, the concentration of components between
saliva and gingival crevicular uid
varies (13) depending on the dierent
physiology of uid formation as well
as dierence in concentration of
markers around the tooth and distance of place of inammation.
Regarding use of the measured biochemical parameters as biomarkers,
our results suggest measured salivary
antioxidants as being accurate for
evaluation of periodontal status and
treatment outcome under applied protocol, as all markers showed clear and
signicant changes in response to

non-surgical periodontal treatment.

However, analysis of correlations
between salivary antioxidants and
clinical parameters showed only two
signicant correlations (GPX/PI and
SOD/GI) where the potential explanation for such a small number of correlations obtained could be that
antioxidants are not suciently concentrated in the saliva to correlate
with periodontal parameters.
Based on obtained results, it
has been shown that periodontal
treatment aects measured salivary
antioxidants, resulting in improvement of TOAC. Simultaneously, our
results suggest salivary TAOC, ALB,
UA, SOD and GPX as reliable biomarkers in evaluating periodontal status and therapy outcome, and saliva
as an accurate experimental uid in
monitoring of periodontal status and
treatment outcome for investigated
biomarkers under the applied protocol. Although promising results have
been obtained in a relatively small
sample, further investigations are
strongly encouraged to obtain and
conrm compatibility among gingival
crevicular uid and salivary levels
under the same homogeneous protocol and increase sample size for more
precise characterization of investigated antioxidants as biomarkers.

Acknowledgements
This study was supported by the Ministry of Education and Science,
Republic of Serbia (project reference
#41008).

References
1. Page RC, Kornman K. The pathogenesis
of human periodontitis: an introduction.
Periodontol 2000 1997;14:911.
2. Cochran DL. Inammation and bone
loss in periodontal disease. J Periodontol
2008;79:15691576.
3. Graves D. Cytokines that promote periodontal tissue destruction. J Periodontol
2008;79:15851591.
4. Kornman K. Mapping the pathogenesis
of periodontitis: a new look. J Periodontol 2008;79:15601568.
5. Chapple ILC. Reactive oxygen species
and antioxidants in inammatory diseases. J Clin Periodontol 1997;24:
287296.

135

6. Meier B, Radeke HH, Selle S et al.

Human broblasts release reactive oxygen species in response to treatment with
synovial uid from patients suering
from arthritis. Free Radic Res Commun
1990;8:149160.
7. McCord JM. Human disease, free radicals, and the oxidant/antioxidant balance. Clin Biochem 1993;26:351357.
8. Gutteridge JMC. Biological origin of free
radicals and mechanisms of antioxidant
protection. Chem Biol Interact 1994;
9:133140.
9. Halliwell B. Oral inammation and reactive species: a missed opportunity? Oral
Dis 2000;6:136137.
10. Waddington RJ, Moseley R, Embery G.
Reactive oxygen species: a potential role
in the pathogenesis of periodontal diseases. Oral Dis 2000;6:138151.
11. Dias IHK, Matthews JB, Chapple ILC,
Wright HJ, Dunston CR, Griths HR.
Activation of the neutrophil respiratory
burst by plasma from periodontitis
patients is mediated by pro-inammatory
cytokines. J Clin Periodontol 2011;38:
17.
12. Battino M, Ferreiro MS, Gallardo I,
Newman HN, Bullon P. The antioxidant
capacity of saliva. J Clin Periodontol
2002;29:189194.
13. Brock GR, Butterworth CJ, Matthews
JB, Chapple ILC. Local and systemic
total antioxidant capacity in periodontitis
and health. J Clin Periodontol 2004;
31:515521.
14. Guentsch A, Preshaw PM, Bremer-Streck
S, Klinger G, Glockmann E, Sigusch
BW. Lipid peroxidation and antioxidant
activity in saliva of periodontitis patients:
eect of smoking and periodontal treatment. Clin Oral Investig 2008;12:345352.
15. Kaklamanos EG, Tsalikis L. A review
on peri-implant crevicular uid assays
potential in monitoring and predicting
peri-implant tissue responses. J Int Acad
Periodontol 2002;4:4959.
16. G
unc
u GN, T
oz
um TF, G
unc
u MB
et al. Myeloperoxidase as a measure of
polymorphonuclear leukocyte response in
inammatory status around immediately
and delayed loaded dental implants: a
randomized controlled clinical trial. Clin
Implant Dent Relat Res 2008;10:3039.
17. Lee JM, Garon E, Wong DT. Salivary
diagnostics. Orthod Craniofac Res
2009;12:206211.
18. Armitage GC. Development of a
classication system for periodontal diseases and conditions. Ann Periodontol
1999;4:16.
19. L
oe H, Silness J. Periodontal disease in
pregnancy. I. Prevalence and severity.
Acta Odontol Scand 1963;21:533551.
20. Silness J, L
oe H. Periodontal disease in
pregnancy. II. Correlation between oral

136

21.

22.

23.

24.

25.

Novakovic et al.

hygiene and periodontal condition. Acta

Odontol Scand 1964;22:121135.
Herrera D, Sanz M, Jepsen S, Needleman I, Roldan S. A systematic review on
the eect of systemic antimicrobials as
an adjunct to scaling and root planing in
periodontitis patients. J Clin Periodontol
2002;29:136159.
Chapple IL, Milward MR, Dietrich T.
The prevalence of inammatory periodontitis is negatively associated with
serum antioxidant concentrations. J Nutr
2007;137:657664.
Panjamurthy K, Manoharan S, Ramachandran CR. Lipid peroxidation and
antioxidant status in patients with periodontitis. Cell Mol Biol Lett 2005;
10:255264.
Borges I Jr, Moreira EA, Filho DW, de
Oliveira TB, da Silva MB, Fr
ode
TS. Proinammatory and oxidative
stress markers in patients with periodontal disease. Mediators Inflamm
2007;2007:45794. doi:10.1155/2007/45794
Tsai CC, Chen HS, Chen SL et al. Lipid
peroxidation: a possible role in the
induction and progression of chronic
periodontitis. J Periodontal Res 2005;
40:378384.

26. Wei D, Zhang XL, Wang YZ, Yang CX,

Chen G. Lipid peroxidation levels, total
oxidant status and superoxide dismutase
in serum, saliva and gingival crevicular
uid in chronic periodontitis patients
before and after periodontal therapy.
Aust Dent J 2010;55:7078.
27. Iwasaki M, Yoshihara A, Hirotomi T,
Ogawa H, Hanada N, Miyazaki H. Longitudinal study on the relationship
between serum albumin and periodontal
disease. J Clin Periodontol 2008;35:
291296.
28. Chapple ILC, Brock GR, Milward MR,
Ling N, Matthews JB. Compromised
GCF total antioxidant capacity in periodontitis: cause or eect? J Clin Periodontol 2007;34:103110.
29. Gustafsson A, Asman B. Increased
release of free oxygen radicals from
peripheral neutrophils in adult periodontitis after Fcg-receptor stimulation. J Clin
Periodontol 1996;23:3844.
30. Sheikhi M, Bouhafs RL, Hammarstrom
KJ, Jarstrand C. Lipid peroxidation
caused by oxygen radicals from Fusobacterium-stimulated neutrophils as a possible model for the emergence of
periodontitis. Oral Dis 2001;7:4146.

31. Sheikhi M, Gustafsson A, Jarstrand C.

Cytokine, elastase and oxygen radical
release by Fusobacterium nucleatumactivated leukocytes: a possible pathogenic factor in periodontitis. J Clin
Periodontol 2000;27:758762.
32. Roche M, Rondeau P, Singh NR,
Tarnus E, Bourdon E. The antioxidant
properties of serum albumin. FEBS Lett
2008;582:17831787.
33. Moore S, Calder KA, Miller NJ,
Rice-Evans CA. Antioxidant activity of
saliva and periodontal disease. Free
Radic Res 1994;21:417425.
34. Tulunoglu O, Alacam A, BaStug M,
Yavuzer S. Superoxide dismutase activity
in healthy and inamed pulp tissues of
permanent teeth in children. J Clin Pediatr Dent 1998;22:341345.
35. Skaleric U, Manthey CM, Mergenhagen
SE, Gaspirc B, Wahl SM. Superoxide
release and superoxide dismutase expression by human gingival broblasts. Eur J
Oral Sci 2000;108:130135.
36. Woodford FP, Whitehead TP. Is measuring serum antioxidant capacity clinically
useful? Ann Clin Biochem 1998; 35
(Pt 1):4856.