**2. Stress response in our body: SAM and HPA system, and possible biomarkers**

In the long history of stress research on human or animals, it has been revealed the existence of two internal stress reaction physiological pathways namely: (1) hypothalamus-pituitaryadrenal (HPA) and (2) sympatho-adrenal-medullary (SAM) system (Kirschbaum, 1994) as shown in Figure 2. When one perceives a stressful situation subjectively, one of or both SAM and HPA system is activated resulting a sort of cascade chain-reaction of a variety of biochemical substances inside our body along with SAM and HPA axes. During such a cascade chain-reaction, wide variety of bio-chemical substances, such as hormones, proteins, and peptides, were released into blood stream or other secretory fluid, such as saliva, urine, breast milk, sweat, etc. Therefore by monitoring such stress-related substances, the activation of SAM and/or HPA system, or nature of perceived stressor is expected to estimate to some extent. This is the background idea.

In the current PNEI studies, mainly seven biomarkers falling under the categories of hormones, immune substances, proteins and enzymes; Immunoglobulin A (IgA), cortisol, human Chromogranin A (CgA), alpha-amylase, Dehydroepiandrosterone (DHEA), Dehydroepiandrosterone sulfate (DHEA-S), and testosterone (TE), has been frequently employed (Deguchi, 2006; Bosch, 2002; Michael, 2000; Nakane, 1999; Kirschbaum, 1994). IgA, CgA, and amylase are considered to reflect SAM activation, and cortisol, DHEA, DHEA-S, and TE are considered to reflect HPA activation. The goal of PNEI study is to investigate the activations of these two systems under various stressful situations using secretory hormones and immune substances, and to demonstrate the congruity of these substances as an objective measurement of human mental stress. In the following contents, these biomarkers are described with reviewing past PNEI studies.

Fig. 2. Stress reaction physiological pathways: HPA and SAM system

#### **2.1 Salivary Immunoglobulin A (IgA)**

248 Biomarker

**DHEA(-S) CgA**

**IgA Amylase Cortisol**

**Testosterone**

**'00 '01 '02 '03 '04 '05 '06 '07 '08 '09**

In the long history of stress research on human or animals, it has been revealed the existence of two internal stress reaction physiological pathways namely: (1) hypothalamus-pituitaryadrenal (HPA) and (2) sympatho-adrenal-medullary (SAM) system (Kirschbaum, 1994) as shown in Figure 2. When one perceives a stressful situation subjectively, one of or both SAM and HPA system is activated resulting a sort of cascade chain-reaction of a variety of biochemical substances inside our body along with SAM and HPA axes. During such a cascade chain-reaction, wide variety of bio-chemical substances, such as hormones, proteins, and peptides, were released into blood stream or other secretory fluid, such as saliva, urine, breast milk, sweat, etc. Therefore by monitoring such stress-related substances, the activation of SAM and/or HPA system, or nature of perceived stressor is expected to

In the current PNEI studies, mainly seven biomarkers falling under the categories of hormones, immune substances, proteins and enzymes; Immunoglobulin A (IgA), cortisol, human Chromogranin A (CgA), alpha-amylase, Dehydroepiandrosterone (DHEA), Dehydroepiandrosterone sulfate (DHEA-S), and testosterone (TE), has been frequently employed (Deguchi, 2006; Bosch, 2002; Michael, 2000; Nakane, 1999; Kirschbaum, 1994). IgA, CgA, and amylase are considered to reflect SAM activation, and cortisol, DHEA, DHEA-S, and TE are considered to reflect HPA activation. The goal of PNEI study is to investigate the activations of these two systems under various stressful situations using secretory hormones and immune substances, and to demonstrate the congruity of these substances as an objective measurement of human mental stress. In the following contents,

Fig. 1. Number of salivary biomarker researches: Retrieved by "PubMed" with queries

**2. Stress response in our body: SAM and HPA system, and possible** 

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**biomarkers** 

"saliva", "stress" and "X (one of each substance)"

estimate to some extent. This is the background idea.

these biomarkers are described with reviewing past PNEI studies.

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100

150

200

250

300

350

An antibody, Immunoglobulin A (IgA), is one of the most important substances in human immune system (Tsujita, 1999). It is present in almost all human secretory fluids, such as saliva, serum, urine, breast milk etc. IgA works non-specifically and therefore plays a very important role on our health, e.g. preventing bacteria from forming colonies, neutralizing toxins and enzymes produced by bacteria, and inhibiting pathogenic viruses from penetrating into the epithelial cell. Especially the salivary IgA called as "the first line of defense" since it functions in the mouse to prevent from contracting influenza or other respiratory tract infection (URTI) illnesses. In fact, clinical studies have suggested the negative correlation between the level of salivary IgA and the incidence of an acute URTI (Jemmott III, 1989). It was also reported the relevance between the level of salivary IgA and caries or periodontitis (Gregory, 1992).

On the other hand, it has been reported that salivary IgA changes its level in response to various psychological factors (Bosh, 2002), such as desirable or undesirable daily events (Stone, 1994), daily hassles (Martin, 1988), negative or positive moods (Martin, 1993), academic stresses such as examination (Jemmott III, 1983) and presentation (Evans, 1994), a short-term stressful cognitive task (Jemmott III, 1989), and relaxation (Green, 1987; Knight, 2001). These stress responses of IgA can be classified into two types: 1) a transient increase of IgA secretion immediately after an exposure of short-term laboratory stressor and subsequent recovering to the basal lever after the removal of the stressor, and 2) decline of basal IgA level several days after or during a long-term stressful situation or under chronic stress. The transient increase of IgA is considered to be regulated by autonomous nervous system (Valdimarsdottir, 1997). Thus it can be taken as a biomarker for SAM system activitiy. The decline of basal IgA level over longer period of time, by contrast, is considered to indicate chronic wear and tear of production capability. Thus it is assumed to represents

Salivary Hormones, Immunes and Other Secretory Substances as Possible Stress Biomarker 251

(Steptoe, 2005). Cortisol has a large diurnal variation of which it starts increase at the just after awakening in the morning, reaches at the peak of the day around 30 minutes after awakening, keeps decreasing through the daytime, and then reaches a minimum level at the night. Especially the individual drastic change, increasing to the peak of the day at the morning is called "cortisol awakening response (CAR)" and it has reported that CAR is associated with chronic stress (Izawa, 2007; Clow, 2004; Schulz 1998; Pruessner, 1997). Altogether salivary cortisol can be a possible short-term and long-term stress marker.

However cortisol has been consistently reported to increase against such stressors accompanying with strong tension and threat, or psycho-social evaluation, it showed inconsistent results against relatively mild stressors such as simple arithmetic task and cognitive task (Dickerson, 2004). Some methodological defeats have been discussed on this discrepancy such as variety of biochemical analysis, saliva collection method, and subject control. Among that, the variety of the timing of saliva sampling has been frequently indicated, including the past PNEI studies with other biomarkers (Hansen, 2008; Dickerson, 2004; Bosch, 2002; Valdimarsdottir, 1997; Kirschbaum, 1994). Also it should be noted that because the stress reaction of HPA system is truly complicated and potentially mediated by variety of physiological factors, the salivary cortisol cannot be taken as a direct measure of

Chromogranin A (CgA), a major member of the granin family of acidic secretory glycoproteins, is known to be released from the adrenal medulla into the blood with catecholamine(Kim, 2005). Therefore it is considered to be a possible biomarker of SAM system (Nakane, 1999; Winkler, 1992). CgA has been reported to transiently increase in response to short-term laboratory stressors such as a calculation test (Nakane, 1999), white noise (Miyakawa, 2006), and a cognitive task (Kanamaru, 2006). On the other hand there are studies failed to observe increase or rather decrease against a laboratory stressors (e.g., Yamakoshi, 2009) and observed a transient increase in subjects who declared the positive

CgA is expected as a possible stress marker representing SAM system activity as just described. However, the number of studies on salivary CgA is still small comparing with that on Cortisol and IgA. Moreover commercial available assay protocol for CgA is limited as just one product (Human Chromogranin A EIA Kit,Yanaihara Institute Inc.). So is

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

HPA system itself but as rather an "indirect" measure (Hellhammer, 2009).

affection after watching a comic video (Toda, 2007).

necessary to accumulate more studies for further discussion.

**2.3 Salivary CgA** 

**2.4 Salivary DHEA** 

context of chronic stress.

some sort of long-term or chronic mental stress state while it is not necessary to consider in relation to HPA system modulation.

Although the transient increase of IgA by laboratory stressors has been consistently observed in the past PNEI studies, the decline of basal IgA over longer period of time according to chronic stress has not been always observed. A review paper on IgA studies pointed out that studies targeting on chronic stress had methodological defects such as less control of subjects' physical conditions e.g. sleep and diet, variety of saliva sampling methods, and introducing non-standardized psychological questionnaires (Bosh, 2002). Moreover the transient increase of IgA would easily mask the change in the basal IgA level over long period of time: if a subject got nervous at the moment of saliva sampling, the observed IgA level would no longer a "basal" level of the IgA of the sampling day.

There are also IgA studies focusing on the effects of various relaxing factors. These studies also reported a transient increase of IgA as the same as studies focusing on laboratory stressors. However few attempts have been made to investigate the effects of such relaxing factors with/under stressful situation (Valdimarsdottir, 1997), so it is not clear whether such a transient increase of IgA induced by relaxing factor would be derived from the same physiological mechanism as the response against laboratory stressors.

Other IgA studies have revealed that the higher cognitive process could mediate the IgA secretion. Psycho-social support alleviated the IgA secretion under long-term (Jemmott III, 1988) and short-term (Ohira, 2004) stressors. Subjects categorized in Type A trait, who are typically represented as short tempered and strong hostile, showed higher baseline of IgA and lower reactivity of IgA against an acute stressor (Ohira, 1999). Controllability of a given stressor unconsciously determined the salivary IgA (Ohira, 2001). These studies suggest that the higher cognitive process could mediate the IgA secretion. However the number of studies targeting on these potential mediators remains small.

Altogether IgA can be a useful stress biomarker, especially for laboratory stressors, representing SAM system activation, while the change in the basal IgA level over long period of time is still be a matter in discussion.

#### **2.2 Salivary cortisol**

Cortisol is the most potent glucocorticoid produced and secreted from adrenal cortex playing a quite important role for maintain our body, e.g., keeping blood glucose level adequately. Cortisol levels can be measured in serum, urine, and saliva. Cortisol is considered to be released into blood stream via activation of HPA system. In addition significant positive correlation has been obtained between salivary and blood cortisol. Therefore salivary cortisol is assumed as a possible stress biomarker representing HPA system activity.

In the past cortisol studies, a transient increase of salivary cortisol was observed by shortterm laboratory stressors, such as mental arithmetic task, stroop task, and oral presentation (Dickerson, 2004; Kirschbaum, 1994). With regard to a long-term or chronic stress, higher level of cortisol secretion has been revealed to associate with chronic stress state, such as job stress (Steptoe, 2000), job loss (Ockenfels, 1995), and divorce (Powell, 2002). By contrast positive affect was found to associate with lower total cortisol secretion during daytime (Steptoe, 2005). Cortisol has a large diurnal variation of which it starts increase at the just after awakening in the morning, reaches at the peak of the day around 30 minutes after awakening, keeps decreasing through the daytime, and then reaches a minimum level at the night. Especially the individual drastic change, increasing to the peak of the day at the morning is called "cortisol awakening response (CAR)" and it has reported that CAR is associated with chronic stress (Izawa, 2007; Clow, 2004; Schulz 1998; Pruessner, 1997). Altogether salivary cortisol can be a possible short-term and long-term stress marker.

However cortisol has been consistently reported to increase against such stressors accompanying with strong tension and threat, or psycho-social evaluation, it showed inconsistent results against relatively mild stressors such as simple arithmetic task and cognitive task (Dickerson, 2004). Some methodological defeats have been discussed on this discrepancy such as variety of biochemical analysis, saliva collection method, and subject control. Among that, the variety of the timing of saliva sampling has been frequently indicated, including the past PNEI studies with other biomarkers (Hansen, 2008; Dickerson, 2004; Bosch, 2002; Valdimarsdottir, 1997; Kirschbaum, 1994). Also it should be noted that because the stress reaction of HPA system is truly complicated and potentially mediated by variety of physiological factors, the salivary cortisol cannot be taken as a direct measure of HPA system itself but as rather an "indirect" measure (Hellhammer, 2009).
