Human salivary alpha-amylase reactivity in a psychosocial stress paradigm

International Journal of Psychophysiology 55 (2005) 333 – 342 www.elsevier.com/locate/ijpsycho

Human salivary alpha-amylase reactivity in a psychosocial stress paradigm Urs M. Natera, Nicolas Rohlederb, Jens Gaaba, Simona Bergera, Andreas Juda, Clemens Kirschbaumb, Ulrike Ehlerta,* a

Institute of Psychology, Clinical Psychology and Psychotherapy, University of Zu¨rich, Zu¨richbergstr. 43, CH-8044 Zu¨rich, Switzerland b Institute of Psychology, Biopsychology, University of Dresden, Germany Received 2 March 2004; received in revised form 20 May 2004; accepted 9 September 2004 Available online 2 November 2004

Abstract Biological indicators for stress reactions are valuable markers in psychophysiological research and clinical practice. Since the release of salivary enzyme alpha-amylase was reported to react to physiological and psychological stressors, we set out to investigate human salivary alpha-amylase changes employing a reliable laboratory stress protocol to investigate the reactivity of salivary alpha-amylase to a brief period of psychosocial stress. In a within-subject repeated-measures design, 24 healthy adults were exposed to the TSST and a control condition on separate days with randomized sequence. Salivary alpha-amylase, salivary cortisol and heart rate were repeatedly measured before, during and after both conditions. Significant differences between psychosocial stress and the rest condition in alpha-amylase activity [ F(3.74,86.06)=4.52; P=0.003], cortisol levels [ F(4.21,88.32)=12.48; Pb0.001] and heart rate [ F(1,22)=81.15; Pb0.001] were observed, with marked increases before and after stress. The data corroborate findings from other studies that showed increased levels of alpha-amylase before and after psychological stress. We discuss the role of salivary alpha-amylase as a promising candidate for a reliable, noninvasive marker of psychosocial stress. D 2004 Elsevier B.V. All rights reserved. Keywords: HPA axis; SAM system; Psychosocial stress test; Salivary alpha-amylase; Cortisol; Heart rate

1. Introduction

* Corresponding author. Tel.: +41 1 6343097; fax: +41 1 6343696. E-mail address: [email protected] (U. Ehlert). 0167-8760/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ijpsycho.2004.09.009

The use of salivary biomarkers has gained increased popularity over the past decade in psychological and biomedical research. While the measurement of free cortisol in saliva has proven

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useful to assess functioning and reactivity of the hypothalamic–pituitary–adrenal (HPA) axis (e.g., Kirschbaum and Hellhammer, 1994), a suitable marker of the sympathoadrenal medullary (SAM) activity in saliva has not been yet found (Schwab et al., 1992; Kennedy et al., 2001). Salivary alphaamylase is a candidate substance to indicate autonomic activity since secretion from human salivary glands occurs in response to neurotransmitter stimulation and salivary glands are innervated by both sympathetic and parasympathetic nerves (Garrett, 1999). Generally, it is considered that sympathetic stimulation [via norepinephrine (NE)] leads to high levels of protein concentrations, e.g., alphaamylase, whereas high rates of fluid output occur in response to parasympathetic cholinergic stimulation [via acetylcholine (ACh); Baum, 1993]. With regard to the secretion of alpha-amylase, the two branches of the autonomic nervous system do not act independently; results from animal studies suggest that both parasympathetic and sympathetic activation lead to an increase in alpha-amylase levels. However, examination of the two branches separately may not be considered a physiological approach to an in vivo situation. In studies combining sympathetic and parasympathetic stimulation, marked elevations in amylase levels have been found (Kyriacou et al., 1988). This methodological approach led to the notion, that amylase secretion is primarily mediated by activation of h1 adrenoceptors (e.g., Schneyer and Hall, 1991). Three major glands (parotid, submandibular and sublingual) and numerous minor glands produce about 500 to 1500 ml of saliva daily. Saliva plays a role in speech and swallowing through its lubricating action, in tasting through its solving action, and in initial enzymatic digestion through one of its major constituents, alpha-amylase (Humphrey and Williamson, 2001). Amylase is produced by the serous acinar cells of the parotid and submandibular glands. It is one of the principal salivary protein appearing as a number of isoenzymes. Amylase accounts for 10– 20% of the total salivary gland-produced protein content and is mostly synthesized by the parotid gland. It is a calcium-containing metalloenzyme that hydrolyzes the a1,4 linkages of starch to glucose and maltose (Zakowski and Bruns, 1985). Alpha-amylase is not only responsible for an initiation of digestion in the oral cavity but it is also considered to play an

important role in binding to oral bacteria (Scannapieco et al., 1993). Due to the insights gained in animal and human research, it was concluded that marked elevations of alpha-amylase concentrations are indicative for autonomic activation. For example, there is evidence that alpha-amylase levels increase in response to physical stressors, such as treadmill exercise (Gilman et al., 1979a), exposure to a high-pressure chamber (Gilman et al., 1979b), running (Nexo et al., 1988; Steerenberg et al., 1997), bicycle exercise (Chatterton et al., 1996; Walsh et al., 1999) or cold exposure (Chatterton et al., 1996). Salivary alpha-amylase levels were also found to respond to psychological stress (Bosch et al., 1996, 1998; Skosnik et al., 2000) or relaxation interventions (Morse et al., 1981a; Morse et al., 1983a,b). While it seems clear that alpha-amylase levels rise following physical stress, the response to a psychological stressor appears to be more inconsistent. This might be due to the psychological nature of the stressors employed or other methodological details. For example, most of these studies measured alpha-amylase levels too infrequently for recording short-term responses to a brief stressor. Since the reported findings on the effects of psychological stress are not consistent in their conclusions, we set out to examine alpha-amylase activity in a standardized psychosocial stress test (Kirschbaum et al., 1993) in a repeated-measures design. Elucidating a detailed course of alphaamylase activity due to psychological stimuli may further establish the use of salivary alpha-amylase as an indicator of human stress reactions.

2. Methods 2.1. Participants Subjects were recruited at the University of Zurich and the Swiss Federal Institute of Technology, Zurich. They received a screening questionnaire, containing exclusion criteria designed to reduce confounding factors that have been shown to affect physiological dependent measures. All subjects were medication-free and refrained from smoking, physical exercise, meals, alcoholic bever-

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ages and low pH soft drinks at least 1 h prior to testing. Subjects with any acute or chronic somatic or psychiatric disorder were also excluded. Since sex differences in endocrine stress responses have been reported (see, e.g., Kirschbaum et al., 1999), only male subjects were included in the present study. After the subjects were provided with complete written and oral descriptions of the study, written informed consent was obtained. The protocol has been approved by the local ethics committee. 2.2. Design and procedure 2.2.1. Psychosocial stress test The Trier Social Stress Test (TSST) has repeatedly been found to induce profound endocrine and cardiovascular responses in 70–80% of the subjects tested (Kirschbaum et al., 1993). After a basal sample of saliva was taken, subjects were introduced to the TSST (2 min). They were then returned to a different room, where they had 10 min to prepare their free speech. Afterwards, subjects were taken back into the TSST room, where they were exposed to a simulated job interview (5 min) followed by a mental arithmetic task (5 min) in front of an audience while standing. Samples of stimulated whole saliva (by chewing on cotton rolls, salivettes) were taken 12 min before, immediately before and after the TSST, with further samples taken at 10, 20, 30, 45 and 60 min to assess alpha-amylase and cortisol. The subjects were told to chew on the salivettes as regularly as possible. Heart rate was assessed continuously during the whole experiment. The TSST was performed between 1400 and 1800 h. 2.2.2. Rest condition The rest condition also took place from 1400 to 1800 h. Each subject was free to choose a quiet activity for spending the 60-min rest period. Magazines and music were made available. During the rest period, alpha-amylase, cortisol, and heart rate was assessed in the same time interval as in the TSST condition. To control for possible sequence effects between the TSST and the rest condition, subjects were randomized into two groups, with group 1 receiving first the rest condition and group 2 receiving first the stress condition.

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2.3. Measures 2.3.1. Sampling methods and biochemical analyses Saliva was collected by the subjects using Salivette (Sarstedt, Sevelen, Switzerland) collection devices. Sampling time was exactly 1 min during which subjects had to chew on the cotton swabs as regularly as possible. Salivettes were stored at 20 8C after completion of the session until biochemical analysis took place. After thawing, saliva samples were centrifuged at 3000 rpm for 5 min, which resulted in a clear supernatant of low viscosity. Fifty microliters of saliva was used for duplicate analyses. Free salivary cortisol was analyzed by using an immunoassay with time-resolved fluorescence detection (Dressendorfer et al., 1992). Intraassay and interassay coefficients of variation were below 10%. To reduce error variance caused by imprecision of the intraassay, all samples of one subject were analyzed in the same run. For alpha-amylase analysis, saliva was also collected with a salivette taken immediately after the salivette for cortisol collection. The devices were stored at 20 8C after completion of the session until biochemical analysis. After centrifugation at 4 8C for 20 min (1800 rpm), saliva was diluted 1:750 in distilled water. Alpha-amylase was analyzed according to Henskens et al. (1996). The activity of alpha-amylase as an indirect measure for alphaamylase concentration was determined using the Sigma reagents bAmylase substrateQ 577-20 and the multienzyme bLin-trolQ M2266 (Sigma, Deisenhofen, Germany). According to the Sigma procedure 577, alpha-amylase hydrolyzes 4,6-ethylideneG7PNP (ET-G7PNP) to G2, G3 and G4 PNP fragments. Alpha-glucosidase hydrolyzes G2PNP and G3PNP to yield p-nitrophenol and glucose. Five moles of substrate (ET-G7PNP) are hydrolyzed to yield 4 mol of p-nitrophenol. p-Nitrophenol absorbs light at 405 nm and following a 2-min lag period, the rate of increase in absorbance is directly proportional to alpha-amylase activity in the sample. Accordingly, 50 Al of saliva was incubated with 150 Al of the substrate (ET-G7PNP) on transparent 96well microtitreplates. After pipetting, plates were brought to 37 8C in a water bath for 90 s. Then, a first absorbance reading at 405 nm wavelength was performed using an anthos HTII microplate reader

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(Anthos Labtech, Salzburg, Austria). The plate was then returned to the water bath at 37 8C for 2 min and the plate was read again at 405 nm. To transfer the increase in absorbance at 405 nm between the first and the second reading into amylase activity expressed as U/ml, the multienzyme Lin-trol (Sigma) was used to establish a standard curve. On each plate, a standard with the following concentrations was run: 785, 638, 478.5, 319, 159.5, 94.2, 31.4 and 0 U/ml. Increases in absorbance of each individual sample were transferred into activity by linear regression using the Curve Expert software (Curve Expert 1.34, Hyams D.G., Starkville, MS, USA). 2.3.2. Electrophysiological measure Heart rate data were obtained via a portable heart rate monitor (Polar Accurex Plus). Heart rate was assessed continuously during both stress and rest period every minute. 2.3.3. Psychometric measures Psychological evaluation of the stressfulness of the stress paradigm was obtained by completion of a visual analogue scale ranging from 0 to 10 with 0 indicating no stress experienced at all. To examine proneness to stress in the subjects, the following questionnaires were used:

2.4. Statistical analysis ANOVAs for repeated measures were computed to reveal possible time and condition effects. 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 randomized groups. Correlations were computed as Pearson product–moment correlations. For alpha-amylase and cortisol, area under the total response curve (AUC), expressed as area under all samples, was calculated using the trapezoid formula (Pruessner et al., 2003). Data were tested for normal distribution and homogeneity of variance using a Kolmogorov–Smirnov and Levene’s test before statistical procedures were applied. With post hoc analysis in the statistical software G-Power (Buchner et al., 1998), we calculated that with the optimal total sample size of N=50, a large effect size of f 2=0.35 with a power of 0.8766 and a=0.05 could have been detected. Since we employed a repeated-measures design, post hoc analysis showed a sufficient sample size. For all analyses, significance level was a=5%. Unless indicated, all results shown are meansFstandard error of means (S.E.M.).

3. Results Trier Inventory for the Assessment of Chronic Stress (TICS2): Perceived chronic stress was assessed. Subjects are required to indicate how often the described stressful situations were experienced during the past year. The TICS2 comprises 10 subscales, namely, dwork overloadT, dsocial overloadT, doverextended at workT, dlack of social recognitionT, dwork discontentT, dsocial tensionT, dperformance pressure at workT, dperformance pressure in communicationT, dsocial isolationT and dworry propensityT (Schulz and Schlotz, 2002). ! Perceived Stress Scale (PSS): A German translation of the Perceived Stress Scale (Cohen et al., 1983) was used to assess the degree to which situations in life experienced during the previous month are perceived as stressful. Items in the PSS were designed to assess how predictable, uncontrollable and overloading participants consider their lives.

!

3.1. Sample characteristics Twenty-five male healthy subjects participated in the study. Randomization resulted in two groups, so that 12 participants underwent the rest condition before the TSST (group 1) and 13 participants underwent the rest period after they performed the TSST (group 2). One participant of group 2 was not able to perform the rest condition due to acute illness. Since we were interested in alpha-amylase reactivity during the stress condition, the subject was included in analyses of the stress condition but not in analyses involving both conditions. The randomized assignment to groups was evaluated by comparing mean age (stress group: 25.31, S.D.=3.3 vs. control group: 23.33, S.D.=2.39, t 23=1.701; P=0.1), and body mass index (stress group: 23.91, S.D.=3.13 vs. control group: 23.12, S.D.=1.99, t 23=0.75; P=0.46). The results of the stress question-

U.M. Nater et al. / International Journal of Psychophysiology 55 (2005) 333–342 Table 1 Psychometric characteristics of all participants Sample data

3.2. Alpha-amylase responses Normative data

Mean S.E.M. Mean S.E.M. PSS

Total score Stress condition Total score Rest condition TICS2 Work overload Social overload Overextended at work Lack of social recognition Work discontent Social tension Performance pressure at work Performance pressure in communication Social isolation Worry propensity

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21.1

6.1

19.62 7.49

19.7

6.1

19.62 7.49

12.4 4.3 5.1 4.1 8.7 4.7 13.9

7.9 2.4 5.4 3.3 5.7 3.0 4.7

13.9 6.1 5.6 6.7 10.6 6.4 13.8

6.3 3.3 3.5 4.0 5.4 3.9 6.1

8.8

2.9

7.9

3.5

5.6 7.6

4.2 5.2

6.9 10.1

4.5 4.7

naires (PSS, TICS2) indicated that the participants were not under significant chronic stress (Table 1). The stress paradigm was evaluated by the subjects as significantly more stressful than the rest condition (visual analogue scale mean TSST: 4.28, S.D.=2.66; mean rest: 0.36; S.D.=0.77; t 24=6.67; P=0.001).

With regard to possible sequence effects, repeated ANOVA was computed including group (group 1– group 2, group 2–group 1) as between-subject factor. Results indicate that group assignment did not influence the responses [ F(7,80.88)=0.43; P=0.89]. The TSST resulted in a significant increase in alpha-amylase activity, as expressed in U/ml [ F(3.6, 82.68)=3.57; P=0.01, see Fig. 1, left], whereas in the rest condition, no significant time effects could be observed [ F(2.56,58.85)=2.32; P=0.09, see Fig. 1, right]. The amount of alpha-amylase activity differed significantly between stress and rest condition [ F(3.74,86.06)=4.52; P=0.003; f 2=0.42]. 3.3. Cortisol stress responses To rule out possible sequence effects, an additional ANOVA was computed. Results show that group assignment did not influence endocrine stress responses [ F(3.71,81.6)=4.3; P=0.35]. As expected, the TSST resulted in a significant cortisol stress response [ F(3.6,79.15)=18.15; Pb0.001; Fig. 2, left]. Groups differed significantly in their endocrine stress response over time [ F(4.21,88.32)=12.48; Pb0.001;

Fig. 1. Salivary alpha-amylase activity in U/ml. Left: a marked increase due to stress may be observed. Right: no significant changes occur in the rest condition.

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Fig. 2. Salivary cortisol levels in nmol/l. Left: a marked increase due to stress may be observed. Right: significant changes over time, possibly due to circadian rhythm, occur in the rest condition.

effect size f 2=1.38; Fig. 2], with subjects in the stress group showing a marked increase in the salivary cortisol response. In addition, Pearson correlations were computed to assess the relationship between salivary alpha-amy-

lase and salivary cortisol levels. Area under the total response curve (AUC) for alpha-amylase in the stress condition was computed. The correlation between the AUC of alpha-amylase and the area under the total response curve of cortisol was not significant

Fig. 3. Heart rate responses in beats per minute (bpm). Left: a marked increase due to stress may be observed. Right: no significant changes occur in the rest condition.

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(r= 0.05; P=0.83, two-tailed). We also computed the net stress reaction for both amylase and cortisol (individual peak measurement minus baseline measurement). The correlation between the two variables was not significant (r= 0.03; P=0.88). 3.4. Heart rate The TSST resulted in a significant heart rate response [ F(5.24,115.3)=8.51; Pb0.001]. There was a significant difference in heart rates between the stress and the rest condition [ F(1,22)=81.15; Pb0.001, see Fig. 3]. In addition, Pearson correlations were computed to assess the relationship between alpha-amylase levels and heart rate response. Correlations were not significant (r=0.16; P=0.46, two-tailed).

4. Discussion The aim of the study was to investigate the reactivity of salivary alpha-amylase in humans in a standardized laboratory stress protocol. Although alpha-amylase activity varied at rest, we were able to demonstrate a significant alpha-amylase stress response to the TSST. The protocol used evoked stress-dependent increases in salivary cortisol and heart rate. No correlations between heart rate, cortisol and alpha-amylase were observed. We were able to show a distinct short-term reaction of salivary alphaamylase activity in a standardized acute psychosocial stress paradigm. The results of some previous studies of alphaamylase reactivity to psychological stimuli have been inconsistent. Our findings seem to be in contrast to studies of Morse et al. (1981b,c, 1982, 1983a). Whereas our results indicate a peak of alpha-amylase activity immediately after an acute stressor, Morse et al. (1981c) found fluctuations in mean salivary protein in an examination paradigm whereas meditation experience altered these fluctuations. Using hypnosis and pharmacological relaxation techniques, the same authors (Morse et al., 1981a) found that these procedures resulted in reduced salivary protein after an endodontic treatment. In another study (Morse et al., 1983b), they were able to show an increase in salivary alpha-amylase after relaxation due to meditation before an endodontic procedure. In contrast to

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the studies of the Morse group, no effect of either stress or relaxation on salivary alpha-amylase activity has been observed in a laboratory situation, using mental arithmetics as a stressor (Borgeat et al., 1984). Bosch et al. (1996) were able to show higher levels of alpha-amylase 30 min before an examination compared with two baselines, 2 and 6 weeks later, respectively. This finding can be explained by anticipation of the stressor beforehand, so that subjects responded physiologically with a rise in alpha-amylase. A similar result was obtained by Chatterton et al. (1997), employing a skydiving paradigm. Subjects preparing for the jump showed significantly elevated alpha-amylase levels immediately before the jump. These somewhat conflicting results could be explained by the different experimental designs used. A previous study found that different stressors with distinct autonomic nervous system effects lead to different outcomes in salivary variables (Bosch et al., 2003). However, only a few of the discussed studies employed a controlled laboratory setting. The amount of measurement points was limited in all the studies, concealing a specific reactive pattern of salivary amylase. In our study, the stressor was employed in a controlled laboratory environment and has repeatedly been shown to elicit strong physiological and psychological stress responses (Kirschbaum et al., 1993). Furthermore, with the employment of random allocation to a rest and a stress condition, intraindividual comparisons have been possible. Finally, with an amount of eight measurement points, we were able to show an accurate reaction pattern of alpha-amylase in a distinct time frame. Taken together, apart from Bosch et al. (2003), this is the only study that has taken all the abovementioned methodological considerations into account. However, whereas Bosch et al. measured amylase secretion rate, we measured amylase concentration indirectly via activity, thus making our results comparable to the abovementioned studies. We did not find significant correlations between the response curves of alpha-amylase and cortisol. This finding is in agreement with Chatterton et al. (1996) who did not find a correlation between the two variables after physiological stress induction (exercise and exposure to heat and cold). This result suggests that alpha-amylase reflects the reaction of a

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different stress system than the HPA axis. But the relationship between alpha-amylase and cortisol should be further examined. There is, however, evidence that salivary alpha-amylase could be an indicator for the activity of the sympathetic–adrenal– medullary (SAM) system. Chatterton et al. have shown significant correlations between alpha-amylase and catecholamines (r=0.64 for norepinephrine and r=0.49 for epinephrine) in physiological stress paradigms (Chatterton et al., 1996). With the possibility of alpha-amylase being an indicator for SAM system changes, it is interesting that alpha-amylase and cortisol (as an indicator for HPA axis changes) are not related, since the SAM system and the HPA axis are closely intertwined (see, e.g., Bugajski et al., 1995; Ziegler et al., 1999). Our study has to deal with possible constraints. Measurement of salivary flow rate is important for the interpretation of changes in salivary protein (Dawes, 1996). Although Bosch et al. (1996) have shown that there is no relationship, neither between alphaamylase output nor activity with salivary flow rate in an intraindividual correlation, the relationship between alpha-amylase activity and flow rate is poorly examined. Further studies should incorporate the measurement of salivary flow rate. Despite this limitation, we hypothesize that the peaks found in our study cannot be ascribed to changes in salivary flow rate, since the changes in alpha-amylase activity were very high due to stress in comparison to the rest condition. However, future studies should empirically corroborate this speculation. Furthermore, no statement can be made whether the observed increase in amylase activity is directly accounted for by an increased secretion of the enzyme or indirectly by changes in salivary flow rate, or even a combination of both. The collection devices we used to obtain saliva are shown to alter the characteristics of several saliva components (Shirtcliff et al., 2001). In that study, however, alpha-amylase was not examined. It could be reasoned that the fluctuations of alpha-amylase in the rest condition are due to the altering characteristics of the salivette devices. However, preliminary results from a collaborative work group suggest that there seem to be no differences in the biochemical characteristics if either cups or salivettes were used (Leicht, unpublished report). Mastication, i.e., chew-

ing on the salivettes, may lead to a higher degree of amylase secretion, since stimulated whole saliva contains a higher proportion of fluid from the parotid gland than does unstimulated saliva (Edgar, 1990). No effect of mastication on alpha-amylase concentration has been found in a study by Mackie and Pangborn (1990), whereas a previous study found, indeed, increased amylase activity due to mastication (Fabian et al., 2003). The results we obtained, however, cannot be explained by this argument. The subjects chewed at eight measurement time points during the stress condition and the rest condition on the collection devices, but it was only in the stress condition (and immediately after completion of this condition) that we could observe marked increases in amylase levels. If amylase activity were increased by mastication, this would have affected alpha-amylase activity in both conditions. In the present study, a marked increase in salivary alpha-amylase activity due to psychosocial stress and a reaction significantly different from a nonstress condition was observed. This corroborates the abovementioned assumption that alpha-amylase can be seen as an interesting parameter in stress research since it is an easily obtained and measured parameter. The underlying mechanism responsible for changes in amylase activity due to psychological stress is not clear from our results. The stressor employed in our study is known to elicit endocrine as well as autonomic neuronal changes. Further studies should investigate the role of parasympathetic and sympathetic activity on increased salivary alpha-amylase levels due to psychosocial stress. To that end, pharmacological blockade and stimulation studies should be conducted. In addition, concomitant measurement of sympathetic and parasympathetic parameters should be incorporated in future studies.

Acknowledgement We gratefully acknowledge the help of Jos A. Bosch for providing details to the amylase assay, and for helpful comments on earlier drafts of this manuscript. We would like to thank Jutta Wolf for laboratory work and Beate Ditzen for help in preparation of the manuscript and Leonie Nicolaus for participating in the gremium of the TSST.

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