Salivary Biomarkers Associated with

Academic Assessment Stress Among

Dental Undergraduates
Vivian Ng, B.Sc. (Hons); David Koh, Ph.D.; Betty Y.Y. Mok, M.Sc.; Sin-Eng Chia, M.D.;
Lum-Peng Lim, Ph.D.
Abstract: This study aims to assess the difference in self-perceived stress before and after an academic assessment and its
association with the marks scored and to study the association of salivary cortisol, Immunoglobulin A (IgA), and chromogranin A
(CgA) with stress levels before and after the assessment. Thirty-one third-year dental undergraduates indicated how stressed they
felt on a 5-point rating scale just prior to and immediately after a one-hour written test. Five-minute unstimulated whole saliva
samples were also collected at those times to measure salivary cortisol, IgA, and CgA. Students rated significantly higher stress
scores before (median [inter quartile range]: 3[1]), compared to after the test (2[2]) (p=0.015). Higher cortisol levels (mean: 6.32
nmol/l) were observed before than after the exam (mean: 5.16 nmol/l) (p=0.015). No significant differences were noted between
the pre- and post-test saliva samples for salivary IgA and CgA levels. A negative correlation was seen between post-test stress
scores and test marks (Spearman’s r = -0.325, p=0.07). Dental students perceived a higher level of stress prior to the test, which
declined after that. Pre-test stress scores were associated with raised salivary cortisol, but not IgA or CgA. Students who
perceived higher levels of stress after the test generally had poorer results.
Ms. Ng is Senior Laboratory Officer and Graduate Student, Dr. Koh is Professor and Head, and Dr. Chia is Associate Professor,
all of the Department of Community, Occupational, and Family Medicine, Faculty of Medicine, National University of
Singapore; Dr. Mok is Senior Lecturer and Dr. Lim is Associate Professor, both of the Department of Preventive Dentistry,
Faculty of Dentistry, National University of Singapore. Direct correspondence and requests for reprints to Dr. David Koh,
Department of Community, Occupational, and Family Medicine (MD3), Faculty of Medicine, National University of Singapore,
16, Medical Drive, Singapore 117597; 65-6874-4988 phone; 65-6779-1489 fax; cofkohd@nus.edu.sg.
Key words: academic examination, self-perceived stress, salivary cortisol, salivary immunoglobulin A, salivary chromogranin A,
dental undergraduates
Submitted for publication 5/13/03; accepted 8/1/03

E
xaminations are the bane of students. How-
ever, they have an important role in evaluat-
ing students’ learning outcomes and their
mastery of a subject. Passing or failing usually has
strong consequences for one’s career development,
so examinations cause psychological stress for most
students.1Academic examinations have been consid-
ered as one of the most acute stresses experienced
by students.2-7
Acute stress has been reported to increase the
activity of the hypothalamus-pituitary adrenal (HPA)
axis with subsequent rise in cortisol level.8 Mucosal
immunity, as reflected by salivary immunoglobulin
A (IgA) levels,9 is also influenced by psychological
stress. With chronic stress, the production of immu-
noglobulin is suppressed, whereas acute psychologi-
cal challenge increases IgA levels. 10 Recently,
Nakane et al.11 and Kanon et al.12 demonstrated that
salivary chromogranin A (CgA) can be a quantita-
tive index for monitoring the activity of the sympa-
thetic nervous system. This constitutes the initial
alarm reaction phase in stress response.
Previous studies have utilized oral presenta-
tions as acute stress situations to study the stress para-
digm.3,5,7 The intensity and nature of the stress en-
countered in an oral presentation might be different
from the stress in a written examination. This study
of dental undergraduate students investigated the use
of salivary cortisol, IgA, and CgA to assess stress
before and after a written term test. Other objectives
of the study were to assess if there was a difference
in self-perceived stress before and after a test and to
study the association of self-perceived stress with
performance on the test.
Materials and Methods
This cross-sectional study was conducted at the
Faculty of Dentistry, National University of
Singapore during a written term test in pediatric den-
tistry. This test is part of the continuous assessment
process that constitutes a small proportion of the stu-
dents’ final examination marks. Students were re-

October 2003 ■ Journal of Dental Education 1091

 quired to answer two essay-type questions in one
hour. Among the topics covered were traumatic in-
juries to anterior teeth in children and management
of problems in the developing occlusion. The test
scripts were all graded by the same staff member.
Ethical approval for the study was obtained from the
Institutional Review Board of National University
Hospital, and informed consent was obtained from
each student. Thirty-one of thirty-two (96.9 percent)
third-year undergraduate dental students participated
in the study. One student was on sick leave. There
were twenty-one males and ten females, aged twenty-
one to twenty-three years (Mean ± SD: 22.7 ± 1.0).
Students were asked to indicate how stressed
they felt on a 5-point scale prior to entry for the writ-
ten test at 11:00 a.m. and again just after they com-
pleted the examination one hour later. The responses
for the question were as follows: 1 = not stressed at
all, 2 = a little stressed, 3 = moderately stressed, 4 =
quite stressed, and 5 = extremely stressed.
In addition, students were requested in advance
not to eat or drink except for water one hour before
saliva collection to minimize possible food debris
and stimulation of salivation. Because this study was
part of a prospective study on salivary biomarkers
and major life events of dental students, students were
familiar with the saliva collection method. Thus,
sample collection should not have imposed any ad-
ditional stress on the students.
A five-minute unstimulated whole saliva
sample was collected immediately before and after
the test. The volume of the saliva was measured and
divided by five minutes to obtain the salivary flow
rate, expressed in ml/min. After the collection, the
samples were kept in an icebox and immediately
transported to the laboratory, where they were stored
at -70˚C until analysis.
Salivary IgA (µg/ml) and cortisol concentra-
tions (nmol/ml) were determined using the
Table 1. Mean values of salivary cortisol, Immunoglobulin A (IgA), and Chromogranin A (CgA) before and after a
written examination among thirty-one dental undergraduates
Salivary Biomarkers Before
Cortisol — Concentration (nmol/ml) 6.32
IgA — Secretion rate (µg/min) 107.6
CgA — Concentration (ρmol/mg protein) 0.43
*Before-After 95% CI refers to the 95% Confidence Interval of difference between before and after test readings.
1092
Salimetrics HS-IgA Kit and HS-Cort Kit, respec-
tively. Both kits were manufactured by Salimetrics
LLC, USA. The concentration of CgA (ρmol/ml) was
measured using the YK070: Chromogranin A (Hu-
man) EIA kit (Yanaihara Institute Inc., Japan). The
Bio-Rad Protein assay kit was used to determine the
protein concentration (mg/ml) in the saliva samples.
Salivary IgA secretion rate (µg/min) was computed
by multiplying the sIgA concentration with salivary
flow rate.3 Because salivary cortisol is not affected
by salivary flow rate, only salivary concentrations
were reported.8 CgA levels were corrected by pro-
tein concentration and expressed as ρmol/mg pro-
tein.11
Data analysis was performed using SPSS 11.0
for Windows. Paired sample t-tests and, where ap-
propriate, non-parametric tests were performed. All
p values and confidence intervals are two-sided, and
the level of statistical significance was set at p<0.05.
Results
The median and inter-quartile ranges of stress
scores before the test were 3 and 1 respectively. Af-
ter the test, the median score was 2, and the inter-
quartile range was 2. The differences were statisti-
cally significant (p=0.015).
The mean cortisol level before the test was 6.32
nmol/l. This was significantly higher than the mean
value of 5.16 nmol/l after the test (p=0.015). Mean
levels for IgA secretion rate and CgA concentration
per total protein were generally higher after than
before the test. However, the differences were not
statistically significant (Table 1).
Generally, stress scores were negatively corre-
lated with test marks. The inverse relationship was
stronger between the post-test self-perceived stress
and test marks (Spearman’s r = -0.325, p=0.07). There
After Before-After 95% CI* P value
5.16 1.01 to 1.50 0.04
119.5 -32.6 to 8.9 0.25
0.45 -0.12 to 0.07 0.69
Journal of Dental Education ■ Volume 67, Number 10
was a poor relationship between pre-examination
perceived stress and examination marks (Spearman’s
r = -0.135, p=0.47).
Discussion
The dental undergraduates perceived a higher
level of stress before than after a written test. This
finding is similar to that of other studies.1,13 The per-
ceived stress of an examination/test may be influ-
enced by the anticipation of difficult examination
questions, feelings, or beliefs regarding mastery in
the subject, relative importance of the exam, and
other factors.
No correlation was observed between the self-
perceived stress scores and any of the salivary
biomarkers (data not shown). A possible explanation
is that the single question used to indicate self-
perceived stress was perhaps too crude a measure to
assess stress. Respondents may underreport or ex-
aggerate the nature of their stress.14 While an in-depth
questionnaire may be useful, it could be argued that
this may impose additional stress on the students.
Cortisol is a major glucocorticoid in humans
that reflects adrenocortical activity. Activation of the
hypothalamic-pituitary-adrenal (HPA) axis and sub-
sequent release of cortisol are major components of
the physiological stress response. Salivary cortisol
accurately reflects serum cortisol, the physiologically
active component.8
Salivary cortisol levels were noted to be higher
prior to the written test. Anticipation of the written
examination is a stressor. Studies have reported in-
creased cortisol levels during anticipation of stress-
ful experiences such as oral academic examinations,
cardiac surgery, and dental treatment procedures.7,15,16
The psychological stress can increase the activity of
the HPA with subsequent rise in cortisol level,8 in-
cluding salivary cortisol.
The concentration and secretion rate of secre-
tory Immunoglobulin A in saliva are good indicators
of the functional status of the mucosal immune sys-
tem. Recent studies on acute stressors such as oral
presentations,17 public speaking,18 and active mental
stress tasks19 showed significant differences in sali-
vary IgA before and after performing the tasks. How-
ever, no significant difference in salivary IgA secre-
tion rates before and after the test was observed in
this study.
It should be noted that the grades for the mid-
term written test in this instance constitute only a
October 2003 ■ Journal of Dental Education
small proportion of the total marks for the subject. It
could therefore be considered a relatively mild stres-
sor in comparison to the final examination faced by
the students. We are aware that the final examina-
tion would be a stronger stressor, which could result
in more significant findings. However, as the final
examination is a very stressful event to students,
many of the students might not wish to participate in
the study, possibly resulting in a poorer response rate.
A study conducted by Lowe et al. on ten final-
year students also showed no significant change in
the salivary IgA levels before and after a written ex-
amination. In contrast, for the same group of stu-
dents, significantly higher levels of salivary IgA were
detected after a thirty-minute oral presentation on
their own academic research project, in front of the
whole class and tutors/assessors.6 As different types
of examinations may induce different intensities of
stress to individuals, changes in salivary IgA may be
sensitive to the intensity of the stressor.
Chromogranin A (CgA) is a 48 kilodalton
acidic glucoprotein that is stored and co-released by
exocytosis with catecholamines from the adrenal
medulla and sympathetic nerve endings.20 Its levels
are correlated with catecholamine release.11
There was no marked difference found in CgA
before and after the test, contrary to the findings of
Nakane et al. who observed a significant elevation
in salivary CgA immediately before an oral presen-
tation in a group of male volunteers.11 Dimsdale et
al. suggested that within the normal physiological
range, which includes situations of mild mental stress,
CgA is stable and slow to respond.20 This could par-
tially explain the lack of an elevated response in this
study, as the midterm test presumably would have a
mild to moderate impact on the stress level of the
students.
An interesting finding was that perception of
higher levels of stress after the examination was
marginally associated with poorer marks for that
exam. This may be due to the awareness of a poor
performance by the student, resulting in an increase
in perceived stress afterwards.
However, both animal and human studies have
indicated that stress and glucocorticoids may impair
memory and cognitive function.21-23 These salivary
biomarkers have the potential of being assessment
tools to identify those students who are consistently
stressed during examinations and who may perform
poorly because of inability to cope with stress. Such
students may benefit from stress awareness educa-
tion and learning of effective coping strategies dur-
1093
 ing the preparation for examinations. However, the
question of how successful such interventions may
be has yet to be answered.
Conclusions
Dental students perceived a level of stress that
was higher before than after the academic assess-
ment. This perception was associated with raised pre-
test salivary cortisol levels but not IgA or CgA. These
findings may suggest that salivary cortisol could be
considered as an indicator for assessing anticipatory
stress. Students who perceived higher levels of stress
after the test generally had poorer results.
Acknowledgments
We wish to thank all the third-year dental stu-
dents at the Faculty of Dentistry, National Univer-
sity of Singapore for their participation in this study.
This study was supported by grant R-186-000-056-
305 from Biomedical Research Council of Singapore.
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Journal of Dental Education ■ Volume 67, Number 10