Salivary alpha-amylase and cortisol responsiveness following electrical stimulation stress in obsessive–compulsive disorder patients

Psychiatry Research ] (]]]]) ]]]–]]]

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Salivary alpha-amylase and cortisol responsiveness following electrical stimulation stress in obsessive–compulsive disorder patients Aimi Kawano, Yoshihiro Tanaka, Yoshinobu Ishitobi, Yoshihiro Maruyama, Tomoko Ando, Ayako Inoue, Shizuko Okamoto, Junko Imanaga, Masayuki Kanehisa, Haruka Higuma, Taiga Ninomiya, Jusen Tsuru, Jotaro Akiyoshi n Department of Neuropsychiatry, Oita University Faculty of Medicine, Hasama-Machi, Oita 879-5593, Japan

a r t i c l e i n f o

abstract

Article history: Received 27 January 2012 Received in revised form 29 July 2012 Accepted 9 November 2012

Salivary a-amylase (sAA) serves as a marker of sympathoadrenal medullary system (SAM) activity. Salivary AA has not been extensively studied in obsessive–compulsive disorder (OCD) patients. In the current study, 45 OCD patients and 75 healthy volunteers were assessed with the Yale–Brown Obsessive–Compulsive Scale (Y–BOCS), the Profile of Mood State (POMS), and the State-Trait Anxiety Inventory (STAI). Measures of heart rate variability (HRV), sAA, and salivary cortisol were also obtained following the application of electrical stimulation stress. The Y–BOCS and POMS Tension–Anxiety, Depression–Dejection, Anger–Hostility, Fatigue, and Confusion scores were significantly increased in patients with OCD compared with healthy controls. In contrast, Vigor scores were significantly decreased in patients with OCD relative to scores in healthy controls. There was no difference in HRV between the patients and the controls. Salivary AA levels in female and male OCD patients were significantly elevated relative to controls both before and after electrical stimulation. In contrast, there were no differences in salivary cortisol levels between OCD patients and controls. The elevated secretion of sAA before and after stimulation may suggest an increased responsiveness to novel and uncontrollable situations in patients with OCD. An increase in sAA might be a characteristic change of OCD. & 2012 Elsevier Ireland Ltd. All rights reserved.

Keywords: Hypothalamic–pituitary–adrenal axis Obsessive–compulsive disorder Salivary a-amylase Salivary cortisol Sympathoadrenal medullary activity

1. Introduction Obsessive–compulsive disorder (OCD) is common psychiatric disorder with a 2%–3% lifetime prevalence worldwide (ValleniBasile et al., 1994; Sasson et al., 1997; Kessler et al., 2005). OCD is characterized by intrusive and inappropriate recurrent thoughts,

Abbreviations: ACTH, Adrenocorticotropic hormone; ANOVA, Analysis of variance; ANS, Autonomic nervous system; CgA, Chromogranin A; Dex/CRH, Combined dexamethasone suppression/ corticotropin releasing hormone stimulation test; DSM-IV, Diagnostic and Statistical Manual of Mental Disorders Fourth Edition; ELISA, Enzyme-linked immunosorbent assay; Gal-G2-CNP, 2-Chloro-4nitrophenyl-4-O-b-D-galactopyranosylmaltoside; HAM-D, Hamilton Rating Scale for Depression; HF, High-frequency; HPA, Hypothalamic–pituitary–adrenal; HRV, Heart rate variability; LC, Locus coeruleus; LF, low-frequency; MDD, Major depressive disorder; OCD, Obsessive–compulsive disorder; SAM, Sympathoadrenal medullary; SNRI, Selective norepinephrine reuptake inhibitors; SNS, Sympathetic nervous system; MINI, Mini-International Neuropsychiatric Interview; POMS, Profile of Mood Statec; S.D., Standard deviation; SNP, Single nucleotide polymorphism; SSRI, Selective serotonin reuptake inhibitors; sAA, Salivary a-amylase; STAI, State-Trait Anxiety Inventory, Y–BOCS, Yale–Brown Obsessive–Compulsive Scale n Corresponding author. Tel.: þ81 97 586 5823; fax: þ 81 97 549 3583. E-mail address: [email protected] (J. Akiyoshi).

impulses or images (obsessions) and repetitive behaviors or mental acts (compulsions) (Goodman et al., 1991). Psychiatric interest has concentrated on the specific interactions between stress and OCD (Sasson et al., 1997). Extensive research has associated OCD with abnormal functioning of the hypothalamic–pituitary–adrenal (HPA) axis. The HPA axis modulates neurohormonal responses to maintain homeostasis in response to stressors (Herman and Cullinan, 1997). Increased activity of the HPA axis in OCD has been reported. Corticotropin releasing hormone (CRH) levels in the cerebrospinal fluid (CSF) have been found to be significantly higher in patients with OCD than in healthy controls (Altemus et al., 1992). Nocturnal adrenocorticotropic hormone (ACTH) and cortisol levels have also been observed to be significantly increased in OCD patients compared with controls (Kluge et al., 2007). Importantly, stress is often a major trigger for the onset of OCD symptoms. Limbic– hypothalamic–pituitary–adrenal (LHPA) axis abnormality has also been reported in patients with OCD. The pituitary gland plays a major role in regulating the body’s physical response to stress. Pituitary volumes were significantly smaller in patients with OCD than in healthy controls (MacMaster et al., 2006; Atmaca et al., 2009). OCD patients who have never been treated with medications have smaller pituitary glands relative to the pituitary glands of

0165-1781/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.psychres.2012.11.010

Please cite this article as: Kawano, A., et al., Salivary alpha-amylase and cortisol responsiveness following electrical stimulation stress in obsessive–compulsive disorder patients. Psychiatry Research (2012), http://dx.doi.org/10.1016/j.psychres.2012.11.010

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A. Kawano et al. / Psychiatry Research ] (]]]]) ]]]–]]]

people who are on medication or individuals without OCD (Jung et al., 2009). Medication may increase the size of the pituitary in people with OCD, and the increase in size may improve regulation of the body’s stress response. There are several reports that deserve mention, e.g. lower responses of the HPA axis to stressors described in subjects with high trait anxiety (Jezova et al., 2004) and in patients with panic disorder (Jezova et al., 2010). Some reports indicate that measurement of salivary a-amylase (sAA) measurement may be informative not only as an indicator of autonomic changes but also as a marker for anxiety reports. Higher adrenaline concentrations have been reported in patients with panic disorder (Jezova et al., 2010), which is consistent with current data. The sympathoadrenal medullary (SAM) system is also associated with anxiety and arousal (Aston-Jones et al., 1994, 1998; Southwick et al., 1999; Berridge and Waterhouse, 2003; Wetherell et al., 2006). It has been suggested that sAA is an index of SAM activity. The basis of this theory is that the sympathetic and parasympathetic branches of the autonomic nervous system innervate the salivary glands. Sympathetic stimulation increases salivary protein secretion, whereas parasympathetic stimulation increases salivary flow rate (Baum, 1993). SAA activity is positively correlated with the acute sympathetic nervous system (SNS) stress response in children and adults (Nater et al., 2006; Gordis et al., 2007). Unlike most salivary analytes, which are actively transported or passively diffused into saliva from plasma (e.g., cortisol and, testosterone), sAA is an enzyme that is locally produced by salivary glands in the oral mucosa. The salivary glands are innervated by sympathetic and parasympathetic nerves, and salivary secretions from various glands (e.g., parotid, submandibular, and sublingual) arise in response to neurotransmitter activation. This suggests that sAA is a prime candidate to measure autonomic activity (Garrett, 1999; Nater et al., 2005). Moreover, studies by Chatterton and colleagues have linked sAA to the SNS component of the stress response (Chatterton et al., 1996, 1997). Only a few studies have failed to observe changes in sAA in response to stressful stimuli including noise (Morrison et al., 2003), the heel prick test in neonates (Schaffer et al., 2008), or a strange situation paradigm (Hill-Soderlund et al., 2008). However, many studies suggest that plasma norepinephrine levels related to the locus coeruleus/autonomic nervous system activity and the stress response in humans can be estimated via the concentrations of sAA. The relationship between OCD and electrical stimuli remains unstudied. Several studies suggest a connection between pain tolerance or sensitivity and emotional distress, such as guilt (Leyro et al., 2010; Bastian et al., 2011). Recently, we reported that sAA and cortisol levels in unremitted patients were significantly elevated compared with levels in controls. Salivary AA levels were significantly correlated with Hamilton Depression Scale (HAM-D) scores in unremitted patients with major depressive disorder (MDD) (Ishitobi et al., 2010). We also reported that sAA levels in female MDD patients were significantly elevated relative to controls both before and after electrical stimulation, while there were no differences in salivary cortisol levels between MDD patients and controls (Tanaka et al., 2012a). We reported that in panic disorder, sAA levels in the alprazolam responder group were significantly elevated compared with the non-responder group and with controls both before and after electrical stimulation (Tanaka et al., 2012b). In this study, we electrically activated the SAM and HPA systems in patients with OCD and examined the resultant biochemical effects. Our hypotheses predicted between-group differences in sAA and cortisol responsiveness. Changes in autonomic measures were expected to parallel changes in sAA and salivary cortisol levels across the groups.

2. Methods 2.1. Participants Participants comprised 45 patients with OCD and 75 healthy controls. Patients were interviewed by a psychiatrist using a semi-functional interview based on the DSM-IV (the Mini-International Neuropsychiatric Interview, MINI). A control group composed of healthy volunteers was recruited from the Oita University Hospital staff. Exclusion criteria included a prior history of OCD incidents, a firstdegree relative with a history of OCD incidents, a body mass index of 32 or greater, the use of steroid-based medications within the past 3 years, and current tobacco use. Subjects were also asked to refrain from eating 3 h before arrival at the hospital and to refrain from taking medication 5 h before arrival. All subjects provided written informed consent following a description of the procedures and risks, and all subjects had the opportunity to ask questions about the study. The study protocol was approved by the Ethics Committee of the Medical Faculty, Oita University, Japan. Patients with a current primary diagnosis of OCD were included. Diagnostic exclusion criteria included: any other mental disorders revealed by the MINI (excluding anxiety disorder) and any acute and/or chronic medical illness as assessed by a physical examination and routine laboratory examination. Patients were carefully matched on these variables with healthy control subjects (except for the use of pharmaceutical drug treatments). A total of 62 patients were approached. Fifteen patients did not meet the inclusion criteria, and two patients dropped out during the procedure. During the interview, the Yale–Brown Obsessive–Compulsive Scale (Y–BOCS) (Goodman et al., 1989) and the Global Assessment of Functioning (GAF) scale were administered. The Y–BOCS is a clinician-administered semi-structured interview and has been regarded as the gold standard for assessing severity of OC symptoms. Its effective use in different ethnic groups has been established (Garnaat and Norton, 2010). The final study sample consisted of 45 patients with a primary diagnosis of OCD, with 11 of these having a secondary diagnosis of panic disorder. The patients were recruited from patients undergoing their first examinations in the psychiatry outpatient department at Oita University Hospital. Three patients also suffered from generalized anxiety disorder. The mean age of onset of OCD was 28.6 (S.D. ¼12.4) years of age. All 45 patients were on various drug treatments at the time of testing, including selective serotonin reuptake inhibitors (SSRIs; n¼ 41) and tricyclic antidepressants (TCA, n¼4). Healthy control subjects (n¼ 75) were recruited at Oita University and were matched by age and gender to the patient sample. Of 186 volunteers, six were excluded due to a current or lifetime major mental disorder as revealed by the MINI. From the remaining 182 volunteers, 75 were selected to provide close matches to the 45 patients with respect to age and gender. Table 1 summarizes the characteristics of patients and matching controls.

2.2. Stimulation Subjects wore stimulator coils, which were connected to a stimulator, on the wrist. This device provided electrical current to the motor and sensory fibers of the median nerve in the right wrist. Subjects were stimulated incrementally until their threshold stimulus, defined as the maximum tolerable stimulus, was reached. We determined the threshold stimulus at the same time that subjects were electrically stimulated for the experiment. To determine the maximum tolerable stimulus, we gave subjects the following instruction: ‘‘If you are not able to bear the pain, please inform us’’. The greatest stimulus lasted 40 s. The whole stimulation period lasted 60–100 s. The mean amplitude of electrical stimulation was between 16 and 22 mA (Table 2). Subjects were told that the level of electrical stimulation would be sufficient to cause pain but would not cause burning or other injury. All participants were tested in the afternoon between 13:00 and 17:00 h. Participants were instructed to refrain from smoking, physical exercise, eating, or drinking caffeinated beverages at least 1 h before testing and after electrical stimulation.

Table 1 Demographic and medical characteristics by group.

N Age (years) Sex (female/male) Comorbidity (%) Y–BOCS

Control

OCD

p or v2

75 32.7 7 6.0 28/47 0(0) 4.8 71.8

45 34.07 14.0 19/26 14 (31.1) 28.17 7.5

0.24 0.49 0.00 0.00

Note. Values are expressed as number, mean (S.D.), or percent Y–BOCS¼ Yale– Brown Obsessive–Compulsive Scale

Please cite this article as: Kawano, A., et al., Salivary alpha-amylase and cortisol responsiveness following electrical stimulation stress in obsessive–compulsive disorder patients. Psychiatry Research (2012), http://dx.doi.org/10.1016/j.psychres.2012.11.010

A. Kawano et al. / Psychiatry Research ] (]]]]) ]]]–]]]

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3. Results

Table 2 Characteristics of major depressive disorder patients and controls. Control (n ¼75)

OCD (n¼ 45)

p

Cormorbidity Panic disorder Generalized anxiety disorder

0 0

11 3

POMS Tension–anxiety Depression–dejection Anger–hostility Vigor Fatigue Confusion

41.8 7 5.6 44.6 7 5.5 43.2 7 6.9 55.3 7 11.7 43.0 7 7.2 42.3 7 7.3

64.0 7 12.8 65.8 7 11.9 50.7 7 13.1 37.4 7 8.3 61.8 7 12.4 65.3 7 13.9

o 0.01 o 0.01 o 0.01 o 0.01 o 0.01 o 0.01

STAI Trait anxiety State anxiety Electrical stimulation

35.4 7 6.8 34.0 7 7.3 23.5 7 16.1

59.1 7 13.1 53.9 7 12.5 13.6 7 13.1

o 0.01 o 0.01 o 0.01

Heart rate valiability LF HF LF/HF

57.2 7 19.8 43.0 7 19.8 2.5 7 4.8

57.5 7 20.6 42.8 7 20.7 2.1 7 2.0

0.48 0.48 0.23

2.3. Procedures We measured sAA and salivary cortisol levels three times in this experiment as in previous reports (Ishitobi et al., 2010). It takes approximately 1–2 min to collect sAA using oral swabs. To examine sAA and salivary cortisol stress responses, we measured sAA and salivary cortisol levels three times (immediately before, immediately after, and 20 min after the intervention). To control for circadian variations in sAA and cortisol levels, the exposure to physical stressors and collection of saliva were performed between 13:00 and 17:00 h (Hammerfald et al., 2006). SAA was measured using the Dry Chemistry System (Nipro Corp., Japan) according to the manufacturer’s protocol. Saliva was sampled by direct immersion of a saliva-sampling strip in saliva under the tongue for 30 s (Robles et al., 2011; Shetty et al., 2011). The strip was immediately placed in an automatic saliva transfer system, and saliva was transferred by compression to the a-amylase test paper on the reverse side of the strip sleeve. The a-amylase test paper contained the substrate 2-chloro-4-nitrophenyl-4-O-b-D-galactopyranosylmaltoside (Gal-G2-CNP). The enzyme reaction started upon transfer by compression and the free CNP level was optically measured after 20 s. The monitor was equipped with a saliva transfer device and an optical device (a light-emitting diode (LED) at a wavelength of 430 nm and a photodetector) (Yamaguchi et al., 2006). The a-amylase activity that reduced sugars equivalent to 1 mmol of maltose was defined as 1 unit. With regard to the reproducibility of the measured results for the saliva transfer volume of the same samples, the coefficient of variation (CV) was 5.5%. A linear regression analysis showed that the slope was 0.0031, and the y-intercept was 0.3524. Within the range of salivary amylase activity between 10 and 140 kU/l, the calibration curve behaved according to the following equation and had a good coefficient with R2 ¼0.97 (Yamaguchi et al., 2006). The concentration of salivary cortisol was analyzed by ELISA (Strickland et al., 2002), with intraassay and inter-assay coefficients of variation of 3% and 10%, respectively. Samples for cortisol measurements were obtained by oral swab. Samples were stored in a freezer at  20 1C until they were thawed for analysis. We recorded high-frequency (HF), low-frequency (LF) and low-frequency/ high-frequency (LF/HF) measures of heart rate variability (HRV) immediately after electrical stimulation by using an APG Heart-Rater SA-3000P (Tokyo Iken Co., Ltd, Japan). We administered the State (STAI-S) and Trait (STAI-T) versions of the Spielberger Anxiety Inventory (Spielberger et al., 1973) and the Profile of Mood States (POMS) to participants before the electrical stimulation protocol (McNair et al., 1992). We also administered the Y–BOCS to patients before electrical stimulation. We administered the three questionnaires 20 min before electrical stimulation.

Regarding POMS testing, the Tension–Anxiety, Depression– Dejection, Anger–Hostility, Fatigue, and Confusion scores were significantly increased in patients with OCD compared with healthy controls (Table 2). In contrast, Vigor scores in patients with OCD were significantly decreased compared with healthy controls. STAI-S and STAI-T scores were increased in patients with OCD compared with healthy controls. The threshold of electrical stimulation applied to OCD patients was significantly lower than that applied to healthy controls (Table 2). There was no difference in any HRV measure between OCD patients and healthy controls (Table 2). There were significant differences in sAA levels between female patients with OCD and healthy controls (F(1, 45) ¼11.32, po0.01, Fig. 1A). There were also significant differences in sAA levels between male patients with OCD and healthy controls (F(1, 71) ¼9.87, p o0.01, Fig. 1B). SAA levels at baseline and after stimulation were both significantly higher in female and male patients with OCD compared with healthy controls. Finally, there were no differences in salivary cortisol levels between female patients with OCD and controls (F(1, 45) ¼ 0.77, Fig. 2A), and there were no differences in salivary cortisol levels between male patients with OCD and controls (F(1, 71) ¼0.17; Fig. 2B). We divided the OCD patients into two groups (checkers, n¼ 36; washers or hoarders, n ¼9). There were no differences in sAA levels between checkers and washers (F(1, 71) ¼1.74). There were also no differences in salivary cortisol levels between checkers and washers (F(1, 71) ¼ 0.16).

2.4. Statistical analysis We adjusted all analyses to match gender and age. The data are presented as the mean 7S.D. of the individual values from each test. We performed analyses using the Statistical Package for the Social Sciences version 19 (SPSS Inc.). We used w2 tests and t-tests for sample characterization. Two-way analysis of variance (ANOVA) was used to compare sAA and cortisol response means, followed by Dunnett’s least significant difference tests. Statistical significance was defined as p o0.05.

Fig. 1. SAA responses to electrical stimulation stress in patients with major depressive disorder and healthy matched control subjects. 1-A: female, 1-B: male. Values are presented as the mean 7 standard error. **p o0.01.

Please cite this article as: Kawano, A., et al., Salivary alpha-amylase and cortisol responsiveness following electrical stimulation stress in obsessive–compulsive disorder patients. Psychiatry Research (2012), http://dx.doi.org/10.1016/j.psychres.2012.11.010

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Fig. 2. Salivary cortisol responses to electrical stimulation stress in patients with major depressive disorder and healthy matched control subjects. 2-A: female, 2-B: male. Values are presented as the mean 7 error.

The stress procedure did not induce any changes in sAA activity or in salivary cortisol concentrations.

4. Discussion In the present study, we demonstrated a strong association between sAA levels and OCD. Before the stimulus exposure, sAA activity was higher in OCD patients compared with activity in controls, but the values did not change throughout the experimental procedure. Salivary AA levels in female and male patients with OCD were significantly elevated compared with controls both before and after electrical stimulation. Salivary AA levels have been proposed as a marker of sympathetic nervous system activity, and sAA levels also appear to be a valid measure of SAM axis activation. The influence of physical stress on sAA levels has been measured in several studies (Chatterton et al., 1996; Nater and Rohleder, 2009). A significant increase in sAA activity was found after exercise (Walsh et al., 1999). Similar results were obtained in another study (Bishop et al., 2006) that examined the influence of caffeine intake on sAA measures in an exercise paradigm. The different sAA and salivary cortisol reactions in response to the physical stressor of taekwondo competition mirrored the faster reactivity of the sympathetic-adrenomedullary system and the slower hypothalamic–pituitary–adrenocortical system, respectively (Capranica et al., 2012). However, O’Donnell et al. (2009) found no significant sAA response to the cold hand stress test, in the morning (11 a.m.) or the afternoon (3 p.m.) in young women. We found significantly higher levels of sAA during this acute stress study

in OCD patients, but not in healthy controls. Our results suggest that patients with OCD might display higher baseline trait and state anxiety even before electrical stimulation. Karadag et al. (2005) also reported that OCD patients had significantly higher trait and state anxiety levels than controls. The STAI assesses trait anxiety (understood as a personality factor that predisposes one to suffer from anxiety) and state anxiety (refers to environmental factors that protect from or generate anxiety). Personality and environmental factors might increase baseline sAA levels. However, our observation of reduced Vigor scores in the OCD group makes this scenario less likely. Several reports support the view that sAA may be a useful indicator for activity of the sympathetic nervous system (Nater and Rohleder, 2009). Salivary AA response can serve as an index for pathological dysregulation of the autonomic nervous system (ANS) in specific clinical and subclinical conditions. Anxietyrelated conditions are associated with autonomic changes (Laederach-Hofmann et al., 2002; Coupland et al., 2003). Salivary AA measurements might provide additional information about autonomic changes occurring in anxiety patients. Some preliminary findings indicate that sAA measurement may be useful not only as an indicator of autonomic changes but also as a marker for anxiety reports. In our study, we found no differences in salivary cortisol levels between patients with OCD and controls before or after stimulation. Regarding the cortisol response to physiological stressors, the tendency toward a lack of response in the OCD patients was surprising, considering their reports of anxiety. One study found no differences in cortisol response to a psychological stressor between patients with anxiety disorder and healthy controls, although neither group showed a significant change (Gerra et al., 2000). A cortisol hyperresponse has been found in samples of phobic patients (Condren et al., 2002; Alpers et al., 2003). This is in sharp contrast to our findings. In a previous report, OCD patients reacted with a near-significant decrease in cortisol response to psychological stressors, while healthy controls reacted with an increase in cortisol (Gustafsson et al., 2008). Because we studied a physiological stressor, the difference in results may be due to methodological issues. Early morning cortisol levels in patients with OCD were significantly elevated compared with a healthy control group, while no significant differences in the late morning or evening measures were observed (Gustafsson et al., 2008). OCD patients had much lower baseline cortisol levels in the morning than controls, but OCD patients had moderately higher baseline cortisol levels in the afternoon compared with controls. Because all participants were tested in the afternoon between 13:00 and 17:00 h, we might not expect to find effects of OCD on baseline cortisol levels. OCD patients and controls in our study also did not differ significantly in their response to electrical stimulation. While our results are partly consistent with other studies (Burke et al., 2005), there might be a circadian effect with respect to OCD and salivary cortisol responsiveness to electrical stimulation. We examined the differences in POMS and STAI scores between the OCD and control groups. We found that the POMS Tension– Anxiety, Depression–Dejection, Anger–Hostility, Fatigue, and Confusion scores were significantly increased in patients with OCD compared with healthy controls, while Vigor scores were significantly decreased in patients with OCD. Our results are consistent with another study in that the POMS Tension–Anxiety and Depression– Dejection scales accurately classified persons with OCD (Gabri¨els et al., 2003). STAI-S and STAI-T scores were increased in patients with OCD compared with healthy controls. These scores were chosen for the assessment of state and trait anxiety. We also examined gender differences in the saliva cortisol and sAA responses before and after electrical stimulation. The sAA

Please cite this article as: Kawano, A., et al., Salivary alpha-amylase and cortisol responsiveness following electrical stimulation stress in obsessive–compulsive disorder patients. Psychiatry Research (2012), http://dx.doi.org/10.1016/j.psychres.2012.11.010

A. Kawano et al. / Psychiatry Research ] (]]]]) ]]]–]]]

levels in both female and male OCD patients were significantly elevated compared with controls before and after electrical stimulation. Some reports suggest that no gender differences exist in the relationship between sAA levels and stress (Filaire et al., 2009; Takahashi et al., 2010), while others suggest that during stressful tasks men have higher sAA levels versus women (van Stegeren et al., 2008). Further studies are needed to examine the relationship between stress and sAA levels in OCD in both genders. Differences might exist between psychological status and hormonal responses. There is no definitive rationale for the observed differences between these groups. One possibility is that the duration of OCD may have affected the results. A second possibility is that individual biological backgrounds of patients with OCD might have affected the results. Specific polymorphisms exert genetic control over stress sensitivity (Cole, 2010; DeRijk et al., 2011). There were no significant differences in HRV values or the strength of applied electrical stimulation between the two groups; thus, biological factors relative to these variables did not influence the differential hormonal responsiveness. It seems that sAA response patterns to physical stressors correspond to the patterns of the sympathetic nervous system. Nater et al. (2006) reported that a psychosocial stress induced a positive correlation between sAA and the low-frequency/high-frequency ratio thought to reflect sympathetic tone. No correlation between plasma norepinephrine and sAA could be established (Nater and Rohleder, 2009). Further studies are needed to examine the relationship between sAA and cardiovascular parameters on exposure to physical stress. A third possibility is that genetic backgrounds in the OCD patients might have affected the results. In this regard, we did not examine genetic differences (e.g., polymorphisms or single nucleotide polymorphisms (SNPs)) between the two groups. However, we intend to examine SNPs in conjunction with stress, HPA axis activity, and SAM axis activity in future studies. Further studies are needed to examine the relationships between hormonal responsiveness, and genetic makeup. This study has four main limitations. First, the number of patients and healthy controls was relatively small, and we will increase the number of participants in future studies. The second limitation is that the number of hormonal examinations was limited. We need to increase the number of hormonal responses tested. We would like to examine salivary chromogranin A (CgA), beta-endorphin, testosterone, and progesterone in future studies. A third limitation is that the OCD patients in this study had been exposed to chronic medication, which may have influenced the results. A fourth concern is that we used the time period immediately preceding the stressor as a baseline. It has been shown that sAA and salivary cortisol levels might differ according to the length of time that a participant spends in the hospital (Balodis et al., 2010). In future studies, we will try to incorporate medication-free patients and perform comparisons between treated and non-treated populations. In conclusion, these preliminary results suggest that increases in sAA might index a characteristic change associated with OCD. As chromogranin A (CgA) is another reliable marker of SAM system activity, it has been suggested that CgA could be used in addition to sAA (Gallina et al., 2011). It seems that our electrical stimulation paradigm did not sufficiently activate the HPA axis, and perhaps further studies should consider the intensity of the applied stressor. Tentatively, we can conclude that the stressor used in this study is not intense enough to stimulate the HPA axis and the SAM system. Additional studies incorporating more frequent measurements and additional combinations of stress markers will be needed to further examine the underlying pathophysiology in patients with OCD.

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Role of funding sources This study was supported by a Grant-in-aid for scientific research (C) from the Ministry of Health and Welfare, Japan.

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Please cite this article as: Kawano, A., et al., Salivary alpha-amylase and cortisol responsiveness following electrical stimulation stress in obsessive–compulsive disorder patients. Psychiatry Research (2012), http://dx.doi.org/10.1016/j.psychres.2012.11.010