**4.1.1 A stressor**

Subjects, ten university male students, were instructed to conduct a simple calculation task as a mild stressor. It was a simple addition of two double-digit integers repeatedly presented on a laptop monitor every 3.0 seconds with changing the figures. It is quite similar to so-called Kraepelin psychodiagnostic test, which is typified as simple, monotonous, and boring arithmetic task, and thus frequently introduced for studies on mental work load or mental fatigue. Subjects were instructed to perform this calculation task as correct and fast as possible.

#### **4.1.2 Procedure**

Two types of experimental design of task/break schedule were prepared for this study as depicted in Figure 4. The experimental session A consists of two sets of 18 minutes of the calculation task and subsequent 9 minutes of the break. The experimental session B consists of six sets of 6 minutes of the task and subsequent 3 minutes of the break. It should be noted that the total duration of the task and break is exactly the same in both sessions: 36 minutes of the calculation task and 18 minutes of the break. After each session A and B, 20 minutes of recovering period was added during which subjects were sitting calmly on the chair.

Saliva samples were taken by a piece of cotton every three minutes during the session. Saliva was also taken at 3, 10, and 20 minutes after the last set of the task/break to observe a

Fig. 4. Task/break schedule in session A and B

relatively mild stressors such as simple arithmetic task and cognitive task by which IgA should always increase (Dickerson, 2004). Actually the idea of "sensitivity of the intensity" cannot stand separately from the "temporal sensitivity." The response of these biomarkers against laboratory stressors always takes place in a transient manner in a certain period of time: it gradually increases, reaches a peak, and after the removal of the stressor, it gradually decreases to the basal level. In fact, if such a transient stress-induced increase of a biomarker took place with a different time delay with respect to each biomarker, it would appear as the difference in the sensitivity of the intensity depending on the time point. Moreover in the case of that saliva sample were not taken frequently enough in the time series, it would result in the inconsistency of obtained experimental results and

With an eye on this point, we designed an experiment to clarify the difference in the sensitivity of biomarkers against a relatively mild laboratory stressor with frequent saliva

The precise changing in the level of salivary four stress biomarkers, which were IgA, cortisol, CgA, and DHEA, were assessed continuously in the time series during which a simple calculation task was given to subjects as a mild stressor. The difference in the sensitivity of these biomarkers as a stress biomarker was expected to be illustrated. Moreover the better understanding for the dynamics of physiological stress response was expected as well since these four biomarkers represent HPA (cortisol and DHEA) and SAM

Subjects, ten university male students, were instructed to conduct a simple calculation task as a mild stressor. It was a simple addition of two double-digit integers repeatedly presented on a laptop monitor every 3.0 seconds with changing the figures. It is quite similar to so-called Kraepelin psychodiagnostic test, which is typified as simple, monotonous, and boring arithmetic task, and thus frequently introduced for studies on mental work load or mental fatigue. Subjects were instructed to perform this calculation task

Two types of experimental design of task/break schedule were prepared for this study as depicted in Figure 4. The experimental session A consists of two sets of 18 minutes of the calculation task and subsequent 9 minutes of the break. The experimental session B consists of six sets of 6 minutes of the task and subsequent 3 minutes of the break. It should be noted that the total duration of the task and break is exactly the same in both sessions: 36 minutes of the calculation task and 18 minutes of the break. After each session A and B, 20 minutes of recovering period was added during which subjects were sitting calmly on the chair.

Saliva samples were taken by a piece of cotton every three minutes during the session. Saliva was also taken at 3, 10, and 20 minutes after the last set of the task/break to observe a

**4.1 Experiment targeting on the difference in the sensitivity among HPA and SAM** 

interpretations among the studies.

**biomarkers against a mild stressor** 

(IgA and CgA) system activities respectively.

**4.1.1 A stressor** 

**4.1.2 Procedure** 

as correct and fast as possible.

collection in the time series, as described in the next.

recovering process of the elevated biomarkers to a basal level. Subjects were instructed to place the cotton under his tongue for three minutes. These cottons were centrifuged at 1500 rpm for 10 minutes to remove mucin. This filtrated saliva samples were stored separately in a small polypropylene tube in freezing chamber at -20 Celsius by the day on the quantitative analysis. Four above-mentioned biomarkers, which are IgA, cortisol, CgA, and DHEA, were assessed by ELISA.

This experiment was conducted as with-in subjects design: every subject went through both sessions A and B in a randomized order. All sessions were conducted in a dark and soundproof room, one by one, and in the afternoon so as to avoid a disturbance by the large diurnal change of biomarkers in the morning.

#### **4.1.3 Other physiological and psychological measures**

Electrocardiogram (ECG) was recorded through all sessions and subsequent 20 minutes of recovering period by a multipurpose bio-signal amplifier at the rate of 500 Hz (BMS-3201, Nihon-Kohden Co.). Frequency analysis was conducted afterward to estimate the high frequency power of the ECG data in the range of 0.15 to 0.40 Hz (hereafter denoted as HF power), which has been frequently used for the index of parasympathetic nervous system activity.

Regarding with the psychological scale, "Profile of Mood State (POMS) (Japanese version)" (Yokoyama, 1993) were given to the subjects to fill up before and after the sessions. POMS is one of the most commonly used questionnaires frequently used various psychological and cognitive science studies (MacNair, 2003). It consists of 65 items concerning subjects' mood state with 5 point scale: not at all, a little, moderately, quite a lot, and extremely. These items are designed to classify into six identified mood factors: tension-anxiety (defined as T-A), depression-dejection (D), anger-hostility (A-H), fatigue-inertia (F), vigour-activity (V), and confusion-bewilderment (C). The score of each mood factors can be found by adding up the scores of corresponding items.

Salivary Hormones, Immunes and Other Secretory Substances as Possible Stress Biomarker 259

respiratory regulated heart beat-to-beat interval modulation, which is so-called Respiratory Sinus Arrhythmia (RSA) (Andreassi, 2007). RSA is dominantly subject to the parasympathetic nervous system, so HF power is considered as an index of the parasympathetic nervous system activities. When one look at the results of our study, it is quite understandable since HF power decreased during the task and recovered in the rest period. Moreover it should be emphasized that such switch-over of the HF power took place rapidly according to the task/break schedule, and well reproduced over time regardless of the repetition of the task/break as seen the session B in particular. Therefore with regard to the "sensitivity" of HF power against a mild stressor, HF power can be compared with a binary switch as it turn on during the task and off in the break without any cumulative

[min]

the mean (S.E.M). Gray background represents a calculation task period.

Fig. 6. Profile of HF power of ECG in session A and B. Error bar represent standard error of

Figure 7 shows the profile of changing in the concentration of salivary secreted IgA during task/break period in the time series in the session A (Fig. 7(a)) and B (Fig. 7(b)). It increased during the task, decreased during the break, and recovered to the initial (basal) level. This simple fact demonstrates the congruity of IgA as a stress biomarker of a mild stressor.

[min]

Fig. 7. Profile of IgA in session A and B. Error bar represent standard error of the mean

IgA Concentration[μg/mL]

(b)

HF Power [msec2]

(b)



[min]

[min]

effect over time.

IgA Concentration[μg/mL]

(a)

**4.2.3 Profile of IgA** 

HF Power [msec2]

(a)



(S.E.M). Gray background represents a calculation task period.

#### **4.2 Results of the experiment: The difference in the sensitivity among HPA and SAM biomarkers**

#### **4.2.1 Behavior and POMS**

The task performance did not show any change by repetition of the task during sessions or between the session A and B. In fact the task, which is the addition of two double-digit integers at every 3.0 seconds, is too easy for the subjects, who were university students, to find any difference in their performance.

Regarding with POMS, only the factor V (vigour-activity) out of six mood factors showed significant decrease after the calculation task (*p*<.05, *t*-test). Other factors did not show any significant change in the scores while the factor F (fatigue) showed a trend of decrease (*p*<.1). In other words, the calculation task introduced in our study was not an intensive stressor which gives subjects negative mood such as tension, threat, or hostility just like TSST but rather a mild stressor as we intended.
