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Salivary -Amylase Levels after Yohimbine Challenge in Healthy Men

Department of Clinical Psychology and Psychotherapy, University of Zurich, CH-8050 Zurich, Switzerland

Address all correspondence and requests for reprints to: Ulrike Ehlert, Ph.D., Department of Clinical Psychology and Psychotherapy, University of Zurich, Binzmuehlestrasse 14, CH-8050 Zurich, Switzerland. E-mail: ehlert{at}psychologie.unizh.ch.

	Abstract

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Context: We and others have previously shown that standardized psychosocial stress significantly increases salivary -amylase (sAA), but it remains unclear whether sAA reflects autonomic nervous system activation.

Objective: The aim of this study was to assess cardiovascular effects and sAA and catecholamine secretion after iv injection of yohimbine.

Design and Setting: We conducted a randomized double-blind placebo-controlled study at an academic research unit.

Participants: Thirteen healthy males (aged 20–28 yr) were examined.

Intervention: Participants received iv injection of yohimbine (0.4 µg/kg) or placebo (0.9% NaCl).

Main Outcome Measures: Eight saliva and blood samples were taken before and after injection for the assessment of salivary flow rate and sAA and catecholamine concentrations. In addition, blood pressure, mood, and anxiety were assessed repeatedly.

Results: Yohimbine induced increases of sAA activity and output in comparison to placebo (P = 0.034). Blood pressure (P < 0.001), salivary flow rate (P = 0.007), and catecholamines (P < 0.001) were also significantly increased. No significant correlations between -amylase parameters and catecholamines were observed.

Conclusions: The results indicate that yohimbine administration activates not only autonomic parameters but also sAA via adrenergic mechanisms, suggesting that sAA might be an indirect indicator of the central sympathetic system.

	Introduction

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SEVERAL STUDIES HAVE supported the assumption that the salivary enzyme -amylase (sAA) is a reliable indicator of physiological and psychological stress. Marked sAA alterations after psychological stressors such as skydiving, an academic examination, or a video task were described by different research groups (1, 2, 3). Chatterton et al. (4) found significant correlations between sAA, epinephrine (E), and norepinephrine (NE) secretion after physical exercise. As a consequence, the authors argued that sAA might be a valid and reliable indicator of (peripheral) catecholaminergic activity.

To establish whether sAA is an indicator of autonomic nervous system (ANS) activation, we exposed healthy men and healthy pregnant and nonpregnant women to a standardized psychosocial stress test and found in all examined groups not only significant cortisol reactions and heart rate acceleration but also sAA increases compared with a rest condition (5, 6). In two additional studies, we tested the hypothesis of an association between sAA release and E and NE secretion after psychosocial stress. We replicated our finding of a stress-dependent increase of sAA. However, we were not able to demonstrate meaningful correlations between sAA, E, and NE (7, 8). Thus, stress-dependent sAA increases do not seem to reflect peripheral catecholamine increases per se but rather changes of the ANS in general. We were able to demonstrate a positive relationship between amylase and sympathetic tone during stress (8), a finding that is corroborated by a recent study on the effects of ß-blockade on sAA, suggesting a direct involvement of ANS mechanisms in the release of sAA (9).

Taking all the findings together, we propose that sAA increases may reflect the interaction of stress-dependent sympathetic and parasympathetic stimulation via central nervous noradrenergic input. To examine this hypothesis, we assessed the indirect effect of yohimbine hydrochloride, an -2-adrenergic receptor antagonist, on sAA release in a randomized placebo-controlled study in healthy men.

	Subjects and Methods

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Study participants

Subjects were recruited through flyers and announcements at the two main universities in Zurich, Switzerland. We recruited healthy, nonsmoking men who were not taking any medication. All eligible individuals expressing an interest in participating in the study were screened using an extensive health questionnaire. All subjects underwent a comprehensive health exam by a physician (G.H.). In addition, a blood sample was drawn to assess a hemogram to exclude individuals with any signs of acute inflammation.

All participants were informed about the course and aim of the study and provided written informed consent before participation. The subjects were remunerated for participation in the study with 100 Swiss francs. The study protocol was approved by the ethics committees of the University of Zurich, as well as of the Canton of Zurich.

Study design

Screening procedures and experiments took place at the laboratory of the Department of Clinical Psychology and Psychotherapy at the University of Zurich. All experimental sessions commenced at 1300 h and lasted for approximately 3 h. All participants were asked to abstain from brushing their teeth for 1 h and to avoid food intake for 2 h before the session. In addition, they were instructed to refrain from exercise, caffeine, and alcohol during the 24 h before the experiment. Under randomized, double-blind, placebo-controlled conditions, each subject received an iv bolus injection of yohimbine hydrochloride (0.4 mg/kg) (Solvay, Hannover, Germany) or 0.9% NaCl at two different sessions with a minimum of 14 d between the two sessions.

Outcome measures

An indwelling forearm catheter was fixed 45 min before yohimbine or NaCl was injected. A total of eight blood samples were taken to analyze E and NE, with two samples drawn before the injection (–20 and –10 min), and the remaining six collected after the injection (+10, +20, +30, +60, +90, and +120 min). At the same time points, eight saliva samples were taken to assess sAA activity and output, and blood pressure was assessed in a supine position. Venous blood was drawn into EDTA-coated monovettes (Sarstedt, Numbrecht, Germany). The catheter line was kept patent by infusion of saline. Immediately thereafter, samples were centrifuged for 10 min at 3000 rpm, with obtained plasma stored at –80 C. Saliva samples were collected after rinsing the mouth with distilled water. Unstimulated whole saliva was collected by accumulating the saliva in the floor of the mouth. After 1 min, saliva was spit into a plastic container and discarded. Collection for sAA analyses lasted for 3 min before saliva was spit into a preweighed plastic vial to measure the weight. sAA output was computed by multiplying salivary flow rate by sAA activity. Samples were stored at –20 C until analysis. Centrifugation of thawed samples was at 3000 rpm for 5 min, resulting in low-viscosity saliva.

Blood pressure was measured using an electronic sphygmomanometer (OMRON-773; Healthcare UK, Milton Keynes, UK).

Mood and anxiety were assessed before and 20 and 60 min after injection, using the German "state version" of the State-Trait Anxiety Inventory (STAI) (10) and a German mood questionnaire [Multidimensionaler Befindlichkeitsfragebogen, (MDBF)] (11). Both questionnaires are widely used and have shown high internal consistency and validity.

Assays

sAA was determined by using the automatic analyzer Cobas Mira and assay kits obtained from Roche. The assay is a kinetic colorimetric test. Intraassay variation for sAA was 1.9%, and interassay variation was 7.4%. E and NE were determined by means of HPLC and electrochemical detection after liquid-liquid extraction (as described in Ref. 12). The detection limit was 10 pg/ml. For quality control during each batch of sample preparation, one control sample (BIO-RAD, level 1) and 23 samples of the study were processed together. The CV for 11 batches was 2.35% for NE (mean, 302 pg/ml) and 4.87% for E (mean, 71 pg/ml).

Data analyses

Physiological and psychological data were analyzed using two-way ANOVA with repeated measurement (group by time). All reported results were corrected by the Greenhouse-Geisser procedure, where appropriate (violation of sphericity assumption). Student’s t tests were computed for comparison of the scale means of the questionnaires with normative samples. Correlations between physiological measures were computed as Pearson product-moment correlations.

Data were tested for normal distribution and homogeneity of variance using a Kolmogorov-Smirnov and Levene’s test before statistical procedures were applied. All tests were two-tailed with significance set at P 0.050.

	Results

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A total of 13 healthy male subjects were enrolled in the study. All men showed regular blood count and no physical complaints or psychiatric disorders. The mean age was 24.9 yr (SD, 2.2), and mean body mass index was 22.6 kg/m² (SD, 1.8).

ANOVAs (group by time effects) showed significant increases under the condition of yohimbine compared with placebo for salivary flow rate (F(3.48/41.73) = 4.38; P = 0.007), sAA activity (F (2.77/ 33.25) = 3.34; P = 0.034) (Fig. 1A), and sAA output (F(3.58/43.01) = 4.30; P = 0.007) (Fig. 1B). Compared with placebo application, yohimbine induced significant increases of E (F(2.88/34.57) = 6.55; P = 0.001) and NE (F(1.92/23.05) = 20.70; P < 0.001) (Fig. 1, C and D). No significant correlations between yohimbine-stimulated sAA (activity and output) and catecholamines were found.

View larger version (27K): [in this window] [in a new window]   FIG. 1. Physiological response to yohimbine/placebo administration. A, Amylase activity in saliva; B, amylase output in saliva; C, E in plasma; D, NE in plasma; E, systolic blood pressure; F, diastolic blood pressure. A–F, Values are given as means ± SEM.

Assessment of mood and anxiety before and after pharmacological challenge revealed that yohimbine significantly increased restlessness (F(1.78/21.30) = 6.83; P = 0.006) and anxiety (F(1.92/ 23.01) = 18.35; P < 0.001) and impaired mood (F(1.61/19.25) = 16.59; P < 0.001) in comparison to placebo, but it did not change the alertness-tiredness scale (F(1.94/23.27) = 2.48; P = 0.107).

	Discussion

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To our knowledge, this is the first study undertaken to investigate the effects of yohimbine challenge on sAA secretion in humans. Our results show that -2-adrenoceptor blockade stimulates not only peripheral NE secretion, which is well known from the literature (13, 14), but also sAA. Because we did not observe meaningful correlations between plasma levels of NE, E, and sAA, we assume that sAA secretion may reflect central NE release instead of peripheral NE secretion. This result is in line with animal findings showing that the release of sAA results mainly from NE released from sympathetic nerves on the primary second messenger cAMP in acinar cells (15). However, NE is not only exerting its influence on the salivary glands; it is also acting on NE receptors throughout the body. NE measured in blood is derived from both adrenomedullary origin and peripheral spill-over. Thus, the weak relationship between sAA and plasma NE might be explained by the differences in origin between central and peripheral NE.

This exciting finding may give rise to new possibilities of ANS measurement. It could make it easier to obtain in-depth insights into psychopathological mechanisms of different psychiatric disorders that are known to be accompanied by autonomic dysregulations. For instance, symptoms of hyperarousal in posttraumatic stress disorder seem to be associated with heightened autonomic arousal and elevated basal and stimulated catecholamine levels (16). Additionally, the personality characteristic of impulsivity, a major symptom of borderline personality disorder, seems to be associated with NE release as shown by Swann et al. (17). The valid and easily assessable method of saliva sampling would allow the depiction of an important central nervous process that appears to be involved in the onset and maintenance of maladaptation to stressful experiences.

	Acknowledgments

 
The opportunity to work in the laboratory of Wolfgang Langhans at the Institute of Animal Sciences, Federal Institute of Technology (Zürich, Switzerland) and the expert technical assistance of Anthony Moses are gratefully acknowledged. Additionally, we thank Dr. Axel Dehn, Clinical Director, Solvay, for manufacturing yohimbine.

	Footnotes

 
Disclosure: U.E., K.E., G.H., and U.N. have nothing to declare.

First Published Online September 12, 2006

Abbreviations: ANS, Autonomic nervous system; E, epinephrine; NE, norepinephrine; sAA, salivary -amylase.

Received March 1, 2006.

Accepted September 5, 2006.

	References

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1. Bosch JA, Brand HS, Ligtenberg TJ, Bermond B, Hoogstraten J, Nieuw Amerongen AV 1996 Psychological stress as a determinant of protein levels and salivary-induced aggregation of Streptococcus gordonii in human whole saliva. Psychosom Med 58:374–382[Abstract/Free Full Text]
2. Chatterton Jr RT, Vogelsong KM, Lu YC, Ellman AB, Hudgens GA 1996 Salivary -amylase as a measure of endogenous adrenergic activity. Clin Physiol 16:433–448[Medline]
3. Takai N, Yamaguchi M, Aragaki T, Eto K, Uchihashi K, Nishikawa Y 2004 Effect of psychological stress on the salivary cortisol and amylase levels in healthy young adults. Arch Oral Biol 49:963–968[CrossRef][Medline]
4. Chatterton Jr RT, Vogelsong KM, Lu YC, Hudgens GA 1997 Hormonal responses to psychological stress in men preparing for skydiving. J Clin Endocrinol Metab 82:2503–2509[Abstract/Free Full Text]
5. Nater UM, Rohleder N, Gaab J, Berger S, Jud A, Kirschbaum C, Ehlert U 2005 Human salivary -amylase reactivity in a psychological stress paradigm. Int J Psychophysiol 55:333–342[CrossRef][Medline]
6. Nierop A, Bratsikas A, Klinkenberg A, Nater UM, Zimmermann R, Ehlert U 2006 Prolonged salivary cortisol recovery in second trimester pregnant women and attenuated salivary -amylase responses to psychosocial stress in human pregnancy. J Clin Endocrinol Metab 91:1329–1335[Abstract/Free Full Text]
7. Rohleder N, Nater UM, Wolf J, Ehlert U, Kirschbaum C 2004 Psychosocial stress-induced activation of salivary -amylase—an indicator of sympathetic activity? Ann NY Acad Sci 1032:258–263[CrossRef][Medline]
8. Nater UM, La Marca R, Florin L, Moses A, Langhans W, Koller MM, Ehlert U 2006 Stress-induced changes in human salivary alpha-amylase activity—associations with adrenergic activity. Psychoneuroendocrinology 31:49–58[CrossRef][Medline]
9. Van Stegeren A, Rohleder N, Everaerd W, Wolf OT 2006 Salivary amylase as marker for adrenergic activity during stress: effect of ß-blockade. Psychoneuroendocrinology 31:137–141[CrossRef][Medline]
10. Laux L, Glanzmann P, Schaffner P, Spielberger CD 1981 Das state-trait-angstinventar. Theoretische grundlagen und handanweisungen. Weinheim, Germany: Beltz
11. Steyer R, Schwenkmezger P, Notz P, Eid M 1997 Der mehrdimensionale befindlichkeitsfragebogen, MDBF (mood questionnaire): Handanweisung. Göttingen, Germany: Hogrefe
12. Ehrenreich H, Schuck J, Stender N, Pilz J, Gefeller O, Schilling L, Poser W, Kaw S 1997 Endocrine and hemodynamic effects of stress versus systemic CRF in alcoholics during early and medium term of abstinence. Alcohol Clin Exp Res 21:1258–1293
13. Murburg MM, Villacres EC, Ko GN, Veith RC 1991 Effects of yohimbine on human sympathetic nervous system function. J Clin Endocrinol Metab 73:861–865[Abstract/Free Full Text]
14. Vythilingam M, Anderson GM, Owens MJ, Halaszynski TM, Bremner JD, Carpenter LL, Heninger GR, Nemeroff CB, Charney DS 2000 Cerebrospinal fluid corticotropin-releasing hormone in healthy humans: effects of yohimbine and naloxone. J Clin Endocrinol Metab 85:4138–4145[Abstract/Free Full Text]
15. Turner RJ, Sugiya H 2002 Understanding salivary fluid and protein secretion. Oral Dis 8:3–10[CrossRef][Medline]
16. Southwick SM, Bremner JD, Rasmusson A, Morgan 3rd CA, Arnsten A, Charney DS 1999 Role of norepinephrine in the pathophysiology and treatment of posttraumatic stress disorder. Biol Psychiatry 46:1192–1204[CrossRef][Medline]
17. Swann AC, Birnbaum D, Jagar AA, Dougherty DM, Moeller FG 2005 Acute yohimbine increases laboratory-measured impulsivity in normal subjects. Biol Psychiatry 57:1209–1211[CrossRef][Medline]

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