**2.4 Salivary DHEA**

Dehydroepiandrosterone (DHEA) and Dehydroepiandrosterone sulfate (DHEA-S) is a steroid adrenal cortex hormone like cortisol. It is thus expected as a possible stress marker representing HPA system activity. It is considered to function antagonistically with cortisol on the central nervous system and immune system (Wolf, 1999). DHEA and DHEA-S was reported to associate with superior stress tolerance (Morgan, 2009). On the contrary lowered DHEA was observed in subjects with partially or completely remitted depression (Michael, 2000). Therefore higher DHEA can be considered in relation with positive state in the context of chronic stress.

Salivary Hormones, Immunes and Other Secretory Substances as Possible Stress Biomarker 253

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

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

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

**0 20 40 60**

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

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

0.0

[min]

0.5

1.0

1.5

**Saliva Volume [mL]**

2.0

2.5

**IgA Saliva**

still at its infancy. Far more research needs to be conducted for more discussion.

methodology has frequently been pointed out as one of a major confounder.

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

**3.1 Saliva sampling method** 

repetitive sampling is not recommended.

**IgA concentration [**

by *Salivette*

**μg/mL]**

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 such as simple arithmetic task and cognitive task is unknown.

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. Further studies are necessary to discuss more.
