**4. Sensitivity in the response of biomarkers against laboratory stressors**

In this section, the transient response of HPA and SAM above-mentioned stress biomarkers against a shot-term laboratory stressor is described with reviewing our past research (Nomura, 2006, 2009, 2010a, 2010b) and also additionally presenting some new experimental results. Especially, the transient response in the time series, i.e. the changing in the level of biomarkers in a short period of time through the onset or the end of an acute laboratory stressor, is featured. It can be highlighting the time constant of the physiological stress response of these biomarkers, or more simply the "temporal sensitivity" against the stressor.

As already described variety of salivary secretory substances are considered to be possible biomarkers as representing the activation of HPA and SAM system. Moreover there seems to be a difference in the "temporal sensitivity" among there biomarkers. Some studies demonstrated the difference in the time course response of these biomarkers against laboratory stressors. Izawa et al. (2008) demonstrated that the stress-induced transient DHEA increase took place ahead of that of cortisol in the TSST experimental session. Ali et al. (2011) also demonstrated in the same TSST session that alpha-amylase also precede cortisol response.

On the other hand whether there would be a difference in the "sensitivity of the intensity" among these biomarkers is still in the matter of discussion. A review article on cortisol described that the salivary cortisol increases against stronger and socially evaluated laboratory stressor, as typified by TSST, but have shown inconsistent results against

Salivary Hormones, Immunes and Other Secretory Substances as Possible Stress Biomarker 257


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

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

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

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

[min]

Task

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

diurnal change of biomarkers in the morning.

**4.1.3 Other physiological and psychological measures** 

Session B

assessed by ELISA.

activity.

scores of corresponding items.

Session A Break Rest

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 interpretations among the studies.

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

#### **4.1 Experiment targeting on the difference in the sensitivity among HPA and SAM biomarkers against a mild stressor**

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 (IgA and CgA) system activities respectively.
