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Anesthesia & Analgesia:
doi: 10.1213/01.ANE.0000184196.60838.8D
General Articles: Brief Report

The Relationship Between Salivary Biomarkers and State-Trait Anxiety Inventory Score Under Mental Arithmetic Stress: A Pilot Study

Noto, Yuka*; Sato, Tetsumi†; Kudo, Mihoko†; Kurata, Kiyoshi‡; Hirota, Kazuyoshi†

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Department of *Nursing, †Anesthesiology, and ‡First Department of Physiology, Hirosaki University School of Health Sciences, Japan

Accepted for publication June 10, 2005.

Address correspondence and reprint requests to Kazuyoshi Hirota, MD, FRCA, Department of Anesthesiology, Hirosaki University School of Medicine, Hirosaki 036–8563, Japan. Address e-mail to

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Measurement of stress hormones is a common objective method for assessment of mental stress. However, the stress of blood sampling alone may also increase stress hormone levels. In the present study, we sampled salivary biomarkers from healthy volunteers under noninvasive conditions and determined their efficacy to assess mental stress. Specifically, we examined the relationship between State Anxiety Inventory score (STAI-s) in subjects exposed to arithmetic stress and salivary chromogranin-A, α-amylase, or cortisol. The STAI-s was significantly correlated to salivary α-amylase (r = 0.589; P < 0.01) but not to salivary chromogranin-A or cortisol. Therefore, salivary α-amylase is a useful indicator of psychosocial stress.

Most surgical patients have preoperative anxiety that is influenced by the uncertainty of anesthesia and surgical procedures, experience, patient’s personality, and coping style (1,2). Preoperative anxiety can have an adverse influence on anesthetic induction and patient recovery (3,4). Thus, anesthesiologists should assess patient’s preoperative anxiety correctly.

Evaluation of preanesthetic anxiety relies on subjective assessment tools such as State-Trait Anxiety Inventory score (STAI) or visual analog scale (5,6). STAI consists of two separate, self-report scales for measuring the distinct concepts of state and trait anxiety (7). State anxiety (STAI-s) can measure the temporary and situational anxiety state accompanied by autonomic excitement. The Trait scale (STAI-t) assesses the tendency of an individual to respond to stressful circumstances under conditions of increased anxiety (7).

However, if patients fail to reliably report, then subjective evaluation tools are of limited value. Therefore, many investigators have sought alternative objective assessment methods. One of the most common objective measures of stress involves an assay of hormone levels. However, blood sampling alone may also increase stress hormone levels by concomitant pain. Recently, measurement of salivary cortisol, α-amylase, and chromogranin-A (CgA), which can be sampled noninvasively, were evaluated as stress biomarkers (8–12).

Salivary cortisol increases with psychological stress (8,13) and correlates serum cortisol (8). The salivary α-amylase is associated with changes in plasma norepinephrine under exercise and psychosocial stress (9). CgA is subject to exocytotic co-release with catecholamines from adrenal medulla and adrenergic neurons (14). Yanaihara et al. (10) found that salivary CgA is a sensitive maker of the initial psychological phase of the stress response. Therefore, these salivary biomarkers may be useful indicators for assessing mental stress. In the present study, we determined whether salivary biomarkers are useful objective indices of mental stress.

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With the approval of our university ethics committee and informed consent, 10 healthy female volunteers (age, 20∼23 yr) participated in the present study. No one had any history of neurological or psychiatric disorders, taking any medication affecting autonomic nervous and endocrine systems, or had any tendency to gingival bleeding. The subjects were required to abstain from eating and drinking except water and brushing teeth 1 and 3 h before the experiment, respectively.

Experiments were performed between 1:00 pm and 6:00 pm to minimize any circadian rhythm effects. After the volunteers had rinsed their mouths with water and an electrocardiogram was monitored, they rested in a chair for 10 min before an experimental session. They were confronted with a mental arithmetic task of 15-min duration. The task required serial subtraction of 13 from a randomly selected 4-digit number shown on a computer screen. Volunteers were instructed to answer verbally at each subtraction. After confirming that the answer was correct, another subtraction of 13 from the correct number of the previous trial shown on the screen was performed.

The anxiety was assessed with STAI-s before and 0 and 10 min after the mental arithmetic task. Simultaneously, saliva was collected with special sampling tubes (Salivettes, Sarstedt, Nümbrecht, Germany). Tubes were then centrifuged for 3 min at 1500g to obtain clear saliva, which was stored at −80°C until assay. Salivary CgA, cortisol, and α-amylase were assayed using kits (YKO70 Human CgA EIA kit; Yanaihara Institute, Shizuoka, Japan; cortisol EIA kit and α-amylase assay kit; Salimeterics Inc., State College, PA, respectively). CgA levels were corrected by salivary protein and expressed as picomole per milligram of protein. Intraassay maximal coefficient of variation was 8.15% for CgA, 6.22% for cortisol, and 6.7% for α-amylase. Interassay maximal coefficient of variation was 12.42% for CgA, 6.88% for cortisol, and 5.8% for α-amylase.

Data are presented as mean ± sd. Statistical analysis was performed using one-way repeated-measures analysis of variance followed by Student-Newman-Keuls test. Correlation between percent change (before the mental arithmetic test = 100%) in STAI-s score and levels of salivary biomarkers was assessed by Pearson correlation coefficient (GraphPad Prism, GraphPad Software Inc., San Diego, CA). A P < 0.05 was considered significant.

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Heart rate (HR) and STAI-s scores increased significantly over the course of the mental arithmetic task and returned to baseline 10 min after completion (Fig. 1). Salivary α-amylase just after completion of the task was significantly higher than that 10 min after the completion and insignificantly higher than that measured before the task. In contrast, salivary CgA and cortisol did not change (Table 1). The STAI-s score significantly correlated with HR and salivary α-amylase (Fig. 2) but not with salivary CgA or cortisol.

Figure 1
Figure 1
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Table 1
Table 1
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Figure 2
Figure 2
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The present data show that the 15-min mental arithmetic task significantly increased both HR and STAI-s score, with a good correlation between the two measures. HR increases by sympathetic excitation. In addition, it has been reported that anxiety positively correlates with HR in women (15). Thus, in the present study, an increase in STAI-s score may reflect mental stress-induced sympathetic excitation.

This is the first report that salivary α-amylase significantly correlated with STAI-s score. Similarly, Takai et al. (16) reported a significant correlation between salivary α-amylase and STAI-t score under psychological stress with stressful video viewing. In addition, Chatterton et al. (9) reported that salivary α-amylase is associated with changes of plasma norepinephrine under exercise and psychosocial stress. Rohleder et al. (17) also demonstrated a positive association between increases of α-amylase and plasma norepinephrine under psychosocial stress. Thus, this biomarker may reflect mental stress.

Although salivary CgA has been reported to reflect stress-induced sympatho-adrenomedullary activity (10,18), salivary CgA was not a good marker in the present study. In agreement with our finding, Ng et al. (19) reported that mental stress induced by academic assessment did not increase salivary CgA. In contrast, Nakane et al. (11) reported that a word processing task significantly increased salivary CgA levels. Our study suggests that a mental arithmetic task may change autonomic tone, but this change may not be strong enough to increase salivary CgA.

Salivary cortisol did not increase by a mental arithmetic task in the present study. Although psychological stressors significantly increase salivary cortisol (13), it should be considered that the major stress response consists of two stages: a short latency catecholamine response in the sympatho-adrenomedullary system and a slower acting cortisol response in the hypothalamus-pituitary-adrenocortical axis (20). Thus, the salivary cortisol response that has a longer latency of secretion seems unsuitable to assess acute stress.

Mental arithmetic task-induced stress may be different from preoperative anxiety. However, both mental arithmetic task-induced stress and preoperative anxiety are classified as psychological stress that activates the hypothalamic-pituitary-adrenal axis and sympathetic-adrenal-medullary system. In addition, a mental arithmetic task is widely accepted as a mental stressor (21,22). Thus, a mental arithmetic task would be a good model to determine the most reliable objective method for assessing psychological stress, including preoperative anxiety.

Most investigators used subjective assessment methods such as STAI score or visual analog scale to evaluate preoperative anxiety (5,6). However, if patients do not report reliably, these subjective evaluation methods are of limited value. Therefore, many investigators have sought alternative objective assessment methods. The measurement of plasma stress hormone concentrations such as catecholamine and antidiuretic hormone are often used as objective assessment for preoperative anxiety (23). However, blood sampling alone increases these stress hormone levels by concomitant pain. In addition, it should be preferable that the measurement of stress hormone concentration can be performed instantly with a portable apparatus. A portable analyzer for salivary α-amylase activity has been developed (24). In the present study, we found that salivary α-amylase activity may reflect psychological stress, although the portable apparatus was not used. Therefore, measurement of salivary α-amylase using the portable analyzer may be a useful objective assessment method for psychological stress, including preoperative anxiety. In conclusion, this study suggests that salivary α-amylase activity may be a good indicator of mental stress.

The authors thank Dr. D.G. Lambert (University Department of Cardiovascular Sciences [Pharmacology and Therapeutics Group], Division of Anesthesia, Critical Care and Pain Management, Leicester Royal infirmary, UK) for his valuable comments.

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