**2.6 Summary of possible biomarkers**

In the current PNEI studies, it is suggested that the salivary biomarkers can be taken as a possible objective stress measurement. All abovementioned substances show a transient increase against short-term laboratory stressor representing the activation of human physiological stress reaction pathways, HPA and SAM systems. Cortisol is the most promising biomarker for a long-term or chronic stress, and IgA is the one for short-term laboratory stressors. However, since PNEI is relatively a new interdisciplinary study, basic problems remain unsolved such as sensitivity of these biomarkers against variety of stressful and relaxing factors, precise change in the secretion of biomarkers in the time series, the effects of long-term stress or chronic stress which might affect the change in the production speed of these biomarkers rather than a temporal change in the secretion.

A recent PNEI research for assessing all different types of biomarkers in blood, such as active natural killer (NK) cell level, varieties of T lymphocyte, dopamine, norepinephrine, and epinephrine, shows a rapid change in the composition of those substances (e.g., Kimura, 2005; Isowa, 2004). It provides quite important information for the better understanding of the human psycho-physiological stress reaction. However such a multi-assessing study is still at its infancy. Far more research needs to be conducted for more discussion.

#### **3. Methods of PNEI Studies using saliva samples**

In this chapter, the methodology of PNEI studies, i.e. experimental designs, subjects' control, preparation of stressors, analysis of biomarkers etc., is described since the variety of methodology has frequently been pointed out as one of a major confounder.

#### **3.1 Saliva sampling method**

252 Biomarker

It was also reported to show a transient increase against short-term laboratory stressors like as other possible biomarkers (Shirtcliff, 2007; Sugaya, 2007; Izawa, 2008). However these studies introduced a strong and socially evaluated laboratory stressor, as typified by "Trier Social Stress Test (TSST) (Kirschbaum, 1993)," responses against relatively mild stressors

Although DHEA in many respects paralleled cortisol secretory activity there was some dissociation in the response in the time series (Sugaya, 2007) and diurnal secretion (Hucklebridge, 2005). DHEA studies are also still small in number as depicted in Fig.1.

Salivary alpha-amylase (sAA) has been reported to show a transient increase against shortterm laboratory stressors along with the activation of sympathetic nervous system mediated via beta-receptor (van Stegeren, 2006; Deguchi, 2006; Yamaguchi, 2004;). Therefore it is also considered to be a possible biomarker representing SAM system activity (Bosch, 2002). However it is suggested that the activation of parasympathetic nervous system also results in a transient increase of sAA mediated via increment of saliva flow rate (Bosch, 2002).

Regarding with chronic stress sAA was reported to have no remarkable association with depression, anxiety, work stress and burnout but a small negative correlation between "social difficulties" measured with a chronic stress scale in a population of nurses (Wingenfeld, 2010). Recently alpha-amylase is used to highlight the difference in the activity of SAM system and HPA system which is measured by salivary cortisol (Strahler, 2010), and a study reported that the salivary alpha-amylase over cortisol ratio, named as AOCg, can be a better indication of stress system dysregulation than sAA or cortisol alone (Ali, 2011).

Free-3-mehoxy-4-hydroxyphenylglycol (free-MHPG) (Okamura, 2010; Buchsbaum, 1981) and testosterone was reported to show transient increase by short-term laboratory stressors, while the number of studies assessing these substances are very small and frequently

In the current PNEI studies, it is suggested that the salivary biomarkers can be taken as a possible objective stress measurement. All abovementioned substances show a transient increase against short-term laboratory stressor representing the activation of human physiological stress reaction pathways, HPA and SAM systems. Cortisol is the most promising biomarker for a long-term or chronic stress, and IgA is the one for short-term laboratory stressors. However, since PNEI is relatively a new interdisciplinary study, basic problems remain unsolved such as sensitivity of these biomarkers against variety of stressful and relaxing factors, precise change in the secretion of biomarkers in the time series, the effects of long-term stress or chronic stress which might affect the change in the

production speed of these biomarkers rather than a temporal change in the secretion.

A recent PNEI research for assessing all different types of biomarkers in blood, such as active natural killer (NK) cell level, varieties of T lymphocyte, dopamine, norepinephrine, and epinephrine, shows a rapid change in the composition of those substances (e.g., Kimura,

such as simple arithmetic task and cognitive task is unknown.

Further studies are necessary to discuss more.

showed inconsistent results (e.g. Schoofs, 2011).

**2.6 Summary of possible biomarkers** 

**2.5 Other salivary possible biomarkers** 

Saliva samples have been collected frequently by "*Salivette*", which is made of dense plain cotton of a cylindrical shape about 1 cm wide and 3.5 cm long. *Salivette* is designed for onetime saliva sampling and mostly introduced in diagnosis uses. In other words, it is not suitable for repetitive saliva collection. It has high absorbability and thus deprives far more amount of saliva, about 2 mL per one sampling for 3 minute, for that of necessary to quantitative determination of biomarkers, at most 50 μL of saliva for one biomarker. Excessive saliva collection brings forth the lack of saliva and, as a result, disturbs normal saliva flow. A past study demonstrated that repetitive saliva collection in every 5 minutes resulted in the decrease of saliva volume and also the concentration of salivary IgA by sampling time as depicted in Fig.3 (Nomura, 2006). Therefore the use of *Salivette* in case of repetitive sampling is not recommended.

Fig. 3. The change in IgA concentration and saliva volume with repetitive saliva collection by *Salivette*

The passive drawing or the use of small cotton is recommended in case of repetitive sampling. Considering the absorption of biochemical substances by the cotton, the passive drawing is technically ideal method for saliva sampling. However it requires training for subjects to get used to dropping saliva into small cup or container. Besides it might be uncomfortable for some subjects to take saliva in this way. Taking saliva by small cotton is easiest method. Although a certain amount of biomarkers would be absorbed, it might be excluded when one focuses on the relative change in the level of biomarkers. It is important

Salivary Hormones, Immunes and Other Secretory Substances as Possible Stress Biomarker 255

materials are included, experimenter can easily assay various biomarkers with a minimum

The principle of ELISA is based on the antigen-antibody reaction which is for capturing a target substance and the enzyme reaction which is for detecting the mass of a target substance via optical density of reaction produced color. The brief description of ELISA (competitive method) is as follows: (1) Thaw saliva samples kept in a biological freezer by moving them into a biological refrigerator (4 Celsius). (2) Centrifuge each saliva samples for 10 minutes at 1500 rpm to precipitate mucins or other solid contents. (3) Add each saliva sample (or known samples for references) into antibody-coated 96-well micro-plate. (4) Add a constant amount of "enzyme conjugate" which is the target biomarker (antigen) combined with horseradish peroxidase (HRP) into the micro-plate and incubate for an hour. In this step, antigen-antibody reaction is occurred competitively between original target in the saliva sample and that in the enzyme conjugate. (5) Wash the micro-plate to flush unbind target. (6) Add tetramethylbenzidine (TMB) solution to induce enzyme reaction with enzyme conjugate which is captured by antigen coated on the bottom of each well of the micro-plate in the step (4). The amount of the bind enzyme conjugate, which is there as a result of competitive reaction process, can be detected as the strength of optimal color (450nm) cased by enzyme reaction. Therefore this optimal density is inversely proportional to the concentration of target containing in the original saliva sample. (7) Finally, the target concentration in each sample is determined by referencing the optimal density of the reference samples. All analysis procedures take roughly about 3 to 5 hours for one microplate. Correct handling of samples and specimens with well calibrated micropipette is

**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

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

knowledge of biochemical analysis.

critical for all steps.

cortisol response.

to prepare the same size and volume of cotton for the repetitive sampling otherwise the volume of saliva collection would become as an unintended confounder. The small cotton should be placed under the subjects' tongue so as to collect the fresh saliva.

Experimenter should pay attention in handling the sample saliva to avoid contingent infection of virus or other possible diseases. All experimenters should ware disposable gloves and glasses. The saliva samples should be kept in the freezer below -20 Celsius by the day of the quantitative determination of biomarkers.
