International Journal of Psychophysiology 90 (2013) 341–346
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International Journal of Psychophysiology journal homepage: www.elsevier.com/locate/ijpsycho
Effects of orthostasis on endocrine responses to psychosocial stress Urs M. Nater a,b,⁎, Beate Ditzen b, Jana Strahler a, Ulrike Ehlert b a b
Department of Psychology, Division of Clinical Biopsychology, Philipps University of Marburg, Germany Department of Psychology, Division of Clinical Psychology and Psychotherapy, University of Zürich, Switzerland
a r t i c l e
i n f o
Article history: Received 12 February 2013 Received in revised form 20 August 2013 Accepted 18 October 2013 Available online 28 October 2013 Keywords: HPA axis SAM system Psychosocial stress Catecholamines Cortisol Orthostasis
a b s t r a c t Standardized psychological procedures have been designed to induce physiological stress responses. However, the impact of standing (orthostasis) on the physiological reaction after psychological stress remains unclear. The purpose of the current analysis was to examine and quantify the relative contribution of orthostasis to the physiological stress response by comparing a “standing with stress” to a “standing without stress” condition. We investigated the effect of standing with and without stress on responses of the sympathetic–adrenomedullary (SAM) system and the hypothalamic–pituitary–adrenal (HPA) axis using a standardized psychosocial stress protocol (Trier Social Stress Test) and a non-stress condition in a repeated measures design. Subjects (N = 30) were exposed to both conditions in randomized order and had to maintain a standing, upright position for 10 minutes. In the “standing with stress” condition, significant increases in repeatedly assessed plasma norepinephrine (NE) and epinephrine (EP), as well as in saliva cortisol were found, while in the “standing without stress” condition, no significant changes in plasma epinephrine and saliva cortisol were observed. Calculations of the relative contribution of orthostasis to physiological stress responses revealed that 25.61% of the NE increase, 82.94% of the EP increase, and 68.91% of the cortisol increase, could be attributed to psychosocial stress adjusted for the effects of orthostasis and basal endocrine output. Although these results are indicative for a marked endocrine reaction that is caused by psychosocial stress alone, our findings show that the contribution of orthostasis must be taken into account when interpreting endocrine data collected in a psychosocial stress test. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The role of stress in everyday life and the detrimental consequences it might exert on our body have been widely examined in the past decades (McEwen, 1998). Assessment of hormonal changes in the two main components of the physiological stress system, i.e. the sympathetic-adrenomedullary (SAM) system and the hypothalamic– pituitary–adrenal (HPA) axis, is essential in psychobiological research to examine the effects of stress and a possible relation to future disease risk (Chida and Steptoe, 2010). Reactivity to psychological stressors in the laboratory has been extensively studied. A plethora of psychological stress paradigms has been developed, albeit with mixed results with regard to endocrine reactivity (Biondi and Picardi, 1999). On the other hand, physiological maneuvers and techniques have been used to evaluate the function of the SAM system (e.g., Valsalva maneuver, cold pressor test, static and dynamic exercise, upright posture (Oribe, 1999)) and the HPA axis (e.g., pharmacological stimulation tests (Heim, Ehlert, and Hellhammer, 2000)). ⁎ Corresponding author at: Department of Psychology, Division of Clinical Biopsychology, Philipps University of Marburg, Gutenbergstr. 18, D-35032 Marburg, Germany. Tel.: +49 6421 24080. E-mail addresses: [email protected] (U.M. Nater), [email protected] (B. Ditzen), [email protected] (J. Strahler), [email protected] (U. Ehlert). 0167-8760/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpsycho.2013.10.010
A frequently cited meta-analysis identified two characteristics of acute psychological stressors and tests that reliably induce changes in HPA axis, i.e. uncontrollability and social-evaluative threat (Dickerson and Kemeny, 2004a). A similar analysis for SAM system changes due to psychological stress is not available. However, research suggests that the SAM reacts more broadly and more sensitively to different challenges, such as physical activity or emotional arousal, than does the HPA axis. As can be seen from the above mentioned stressors used to stimulate the SAM system, it is of pivotal importance to have information about how much of the variance is contributed by psychological characteristics (e.g., uncontrollability) and how much of the variance of the effect can be attributed to physical characteristics of the stressor (e.g., standing). In the laboratory, exposure to psychological stressors is usually studied with subjects in a sitting position. However, the simulation of real-life situations, especially when examining the effects of psychosocial stress on human subjects, demands a more realistic setting. Thus, some stress tests have been conducted with the subjects being examined in an upright, standing position (e.g. Kirschbaum, Pirke, and Hellhammer, 1993). Furthermore, subjects are also often required to walk between different rooms in a laboratory (i.e. resting and testing room). Besides these well-established stress tests (Dickerson and Kemeny, 2004a; Gaab et al., 2003; Roy, 2004; Singh, Petrides, Gold, Chrousos, and Deuster, 1999), effects of orthostasis on parameters of the SAM system and the HPA axis have been observed (Bie, Secher,
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Astrup, and Warberg, 1986; Matzen, Secher, Knigge, Bach, and Warberg, 1992; Vlcek, Radikova, Penesova, Kvetnansky, and Imrich, 2008). Assuming the upright posture leads to sharp rises in catecholamine concentrations, especially in norepinephrine (NE). In healthy subjects, changing posture from lying to sitting results in higher concentrations of NE, and in epinephrine (EP) (Cameron et al., 1987), and lying vs. walking increases NE, but not EP concentrations (Robertson et al., 1979). Changing from lying to standing results in higher NE levels (Christensen and Brandsborg, 1976; Cryer, Santiago, and Shah, 1974; Lechin et al., 1995a; Lechin et al., 1995b; Paramore, Fanelli, Shah, and Cryer, 1998; Schofl, Becker, Prank, von zur Muhlen, and Brabant, 1997; Vlcek et al., 2008), whereas in EP, the results are inconclusive: some studies found higher concentrations (Paramore et al., 1998; Schofl et al., 1997; Vlcek et al., 2008), others found no changes (Christensen and Brandsborg, 1976; Cryer et al., 1974; Lechin et al., 1995a; Lechin et al., 1995b). Finally, changing from sitting to standing leads to increased NE and EP (Tulen, Boomsma, and Man in 't Veld, 1999). Orthostatic challenge (via tilt) also results in a rise of other physiological parameters, such as heart period and blood pressure (Hatch, Klatt, Porges, Schroeder-Jasheway, and Supik, 1986). With regard to HPA axis activation, increases in cortisol when changing from a sitting to a standing position have also been found. Plasma cortisol was shown to rise in response to 40 minutes of standing (Abalan et al., 1992). The same was true for salivary cortisol in a balanced cross-over design examining the effect of 20 minutes of sitting, lying, or being in the upright posture with increasing values only in the upright condition (Hennig et al., 2000). However, there are also studies lacking any evidence of postural shift affecting salivary cortisol (Hucklebridge, Mellins, Evans, and Clow, 2002). These findings of postural changes stimulating the SAM system and the HPA axis make it essential to consider orthostasis-induced changes in the respective systems when examining psychosocial stress effects. Studies investigating healthy subjects in either sitting or standing positions or those which require walking from one room to the next, thus risk a possible confounding effect by orthostasis. It might very well be that the observed results are just due to standing or walking rather than due to the psychosocial or mental stress elicited by the task. Addressing the possible confounding effect of orthostasis, Tulen et al. (1999) found no differences in plasma catecholamines between standing and sitting for subjects doing the word–color–Stroop test. There is also evidence that during another well-examined standardized psychosocial stress test, the Trier Social Stress Test (Kirschbaum et al., 1993), postural changes have an impact on autonomic measures. Research indicates that asking the participants to assume an upright position for 10 minutes as a control condition results in heart rate and heart rate variability changes comparable to the TSST condition (Rohleder, Wolf, Maldonado, and Kirschbaum, 2006). In this study, salivary cortisol was unaffected by postural changes during the control condition. Replicating and extending this finding, our group was able to show an effect of standing during such a control condition on NE while EP seemed to be unaffected by postural changes (Nater et al., 2006). These findings were confirmed by a third TSST study that showed increases in heart rate, blood pressure, NE, aldosterone and renin activity to postural changes, while ACTH, cortisol and EP remained unaffected (Mlynarik, Makatsori, Dicko, Hinghofer-Szalkay, and Jezova, 2007). Furthermore, changes of serum cholesterol levels were shown in response to mental stress and standing, but controlling for body shift-induced changes in hemoconcentration diminished the effect of both tasks (Muldoon et al., 1992). The combined effect of postural and mental stress on hemoconcentration was examined in a well-designed study on the effects of head-up-tilt and a mental stressor both alone and in combination on rheological and cardiovascular measures. Changes in blood pressure, total peripheral resistance, plasma volume and hematocrit were highest in the combined condition while cardiac responses did not differ from those to postural stress alone (Veldhuijzen Van Zanten, Thrall, Wasche, Carroll, and Ring, 2005).
In this present work, we will extend previous work by calculating the amount by which psychosocial stress will change endocrine (i.e. catecholamines and cortisol) activity in comparison with changes in body posture. The findings from this study will have implications for the assessment of the relative contribution of orthostasis on psychosocial stress responses and might guide future research in choosing a study design that may take into account orthostasis as a potentially confounding factor. We studied the influence of standing on both catecholamines and cortisol in a balanced exposition to a stress and a no-stress condition in a repeated measures design. This procedure allows us to separate the physiological component and the psychological component in a psychobiological stress paradigm. It is hypothesized that EP and cortisol would not significantly change in response to the “standing without stress” compared to the “standing with stress” condition, while NE is expected to increase in both conditions. It is further expected that a considerable percentage of the change of endocrine markers is just due to postural shift. 2. Methods and materials 2.1. Participants Subjects were recruited through advertisement at the University of Zurich and the Swiss Federal Institute of Technology, Zurich to participate in a larger project on the effects of psychosocial stress on various stress markers. Stress effects on salivary cortisol and α-amylase, plasma catecholamines, and heart rate variability have been previously reported (Nater et al., 2006). Thirty healthy male subjects participated in the study twice, during a “standing with stress” and during a “standing without stress” condition (for subject characteristics see Table 1). All subjects were medication-free and were non-smokers. Subjects with any acute or chronic somatic or psychiatric disorder (as evidenced by self-report) were excluded from the study. Participants were told not to perform any strenuous physical activity 48 hours prior to the experiment and to cease all sporting activities during the time of the study. Intake of ethanol and caffeine was forbidden 18 hours prior to the experiment. At least 60 minutes before the study, subjects were not allowed to eat or brush their teeth so as to avoid gingival bleeding. After the subjects were provided with complete written and oral descriptions of the study, written informed consent was obtained. The subjects were remunerated for participation in the study with 80 Swiss Francs. This study adhered to human experimentation guidelines of the Helsinki Declaration. The study protocol was formally approved by the ethics committee of the University of Zurich and the ethics committee of the Canton of Zurich. 2.2. Procedures All subjects were exposed to a standardized control condition (“standing without stress”) and the Trier Social Stress Test (TSST) (“standing with stress”) with a gap of 2 weeks (±3 days) in between.
Table 1 Subject characteristics and AUCG values. N = 30
“Standing with stress”
“Standing w/o stress”
Age in years Height in cm Weight in kg BMI in kg/m2 AUCG cortisol AUCG epinephrine AUCG norepinephrine
24.8 ± 2.4 182.3 ± 7.7 74.8 ± 9.2 22.5 ± 2.0 518.04 ± 261.68 1 565.17 ± 902.83 12 706.58 ± 4 963.14
306.70 ± 246.86 855.58 ± 542.04 10 116.25 ± 2 840.25
Abbreviations: BMI = body mass index; AUCG = area under the curve with respect to ground (calculated using the trapezoid formula, see Pruessner et al. (2003)).
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Subjects were randomized to either receive the control condition first, or to receive the stress condition first, in order to control for possible sequence effects. The TSST consists of a simulated job interview (5 minutes) followed by a mental arithmetic task (5 minutes) in front of an audience. This task has repeatedly been found to induce profound endocrine changes in 70–80% of the subjects tested (Kirschbaum et al., 1993). In both conditions, an indwelling catheter was inserted into the antecubital vein of the non-dominant arm after arrival. After a baseline of 40 minutes, the experiment started. In both the stress and the control condition, subjects had to change from sitting into the upright, standing position, and remain standing for 10 minutes. Saliva and blood samples were taken 10 minutes before (−10 minutes), immediately before (0 minutes), 5 minutes after beginning (+5 minutes), and directly after the stress test (+15 minutes), with a further sample taken 10 minutes after the stressor (+25 minutes). One additional saliva sample was taken 20 minutes (+35 minutes) after the stressor had ended to capture the delayed reaction of salivary cortisol. All experiments were performed between 2 p.m. and 6 p.m. to control for diurnal variation in endocrine parameters. 2.3. Endocrine assessment Blood samples were taken with EDTA-coated monovettes (Sarstedt, Sevelen/Switzerland), and kept on ice until centrifugation at 3000 rpm for 10 minutes. Six hundred microliters was pipetted in pre-cooled aliquots. Samples were stored at −80 °C until assayed. Plasma norepinephrine and epinephrine were determined by means of HPLC and electrochemical detection after liquid–liquid extraction (as described elsewhere, Ehrenreich et al., 1997). Saliva was collected using Salivette® collection devices (Sarstedt, Sevelen/Switzerland) and stored at −20 °C after completion of the session until biochemical analysis took place. After thawing, saliva samples were centrifuged at 3000 rpm for 5 minutes. Salivary free cortisol was analyzed by using an immunoassay with time-resolved fluorescence detection (Dressendorfer, Kirschbaum, Rohde, Stahl, and Strasburger, 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.
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3. Results The subject characteristics can be found in Table 1. As previously reported, the TSST resulted in significant increases in all hormones (plasma norepinephrine and epinephrine, and salivary cortisol). Comparing those responses to the control condition (“standing without stress”) revealed significant differences between both conditions (for further details please refer to Nater et al., 2006). There were no statistically significant increases in the control condition for EP (Ftime (2.84/85.3) = 1.52; p = 0.22) and salivary cortisol (Ftime (1.39/41.79) = 2.42; p = 0.12), while NE was significantly increased during the control condition (Ftime (2.69/ 80.75) = 39.18; p b 0.001). The areas under the curve with respect to the ground (AUCG) for all parameters were computed (see Table 1). Results show significantly higher concentrations of all parameters during the “standing with stress” condition in comparison to the “standing without stress” condition (AUCG for NE: t29 = 2.98; p = .006; EP: t29 = 5.59; p b .001, cortisol: t28 = 4.66; p b .001). There was no effect of allocation order on AUCG values (NE: F[1,28] = 2.56, p = 0.121; EP: F[1,28] = 0.18, p = 0.677; cortisol: F[1,27] = 0.12, p = 0.737). There was an effect of allocation order on baseline cortisol values on the “standing with stress” day with higher values in those being allocated to this condition first (t28 = 2.37, p = 0.025). No effect was found for baseline values on the “standing without stress” day and for NE and EP values (all p N 0.162). Adding this cortisol baseline value of the stress condition as a covariate, the difference between both conditions was no longer significant (F[1,27] = 0.46, p = 0.502). For all parameters, the relative contribution of standing was analyzed by computing percent of change in the AUCG between the “standing with stress” condition and the “standing without stress” condition on a within-subject basis. The AUCGs in the “standing with stress” condition are 12706.58 pg/ml (NE), 1565.17 pg/ml (EP), and 518.04 nmol/l (cortisol). The change in percentage in the “standing with stress” condition relative to the “standing without stress” condition was 25.61% for NE, 82.94% for EP, and 68.91% for cortisol representing the specific variance of endocrine activity that can be attributed to psychosocial stress adjusted for the effects of orthostasis and basal endocrine output (please see Figs. 1 to 3 for an illustration of these findings).
2.4. Statistical analysis Analyses of variance (ANOVAs) for repeated measures were computed to reveal possible time × condition interactions. All reported results were corrected by the Greenhouse–Geisser procedure where appropriate (violation of sphericity assumption). For catecholamines and cortisol, area under the total response curve with respect to the ground (AUCG) was calculated using the trapezoid formula, according to 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. Due to violation of the normal distribution of some AUCG values for cortisol, norepinephrine, and epinephrine (analyzed by the Kolmogorov–Smirnov test), these measures were log-transformed. Student's t-tests and univariate ANOVAs were computed for comparison of endocrine output between the two conditions. To analyze the relative contribution of psychosocial stress, the percent of change in AUCG between the “standing with stress” condition and the “standing without stress” condition was computed (% change = [AUCG“standing with stress” − AUCG“standing without stress”] / AUCG“standing without stress” * 100) representing the specific variance of total endocrine activity that can be attributed to psychosocial stress. The variance due to changes in body posture and basal activity, taken into account by using AUCG, is thus represented in 100 − % changestress. One further advantage of relating the total output during both conditions is to also control for basal endocrine activity which is included in AUCG. For all analyses, significance level was α = 5%. Unless indicated, all results shown are means ± standard deviation (SD).
Fig. 1. Salivary cortisol during “standing with stress” and “standing without [w/o] stress” condition. Time of standing is indicated by the black bar. In the “standing with stress” condition, the black bar also indicates the time of the stress test. Error bars indicate SEM. The shaded area illustrates the increase of cortisol that could be attributed to psychosocial stress adjusted for the effects of orthostasis and basal output.
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Fig. 2. Plasma norepinephrine during “standing with stress” and “standing without [w/o] stress” condition. Time of standing is indicated by the black bar. In the “standing with stress” condition, the black bar also indicates the time of the stress test. Error bars indicate SEM. The shaded area illustrates the increase of norepinephrine that could be attributed to psychosocial stress adjusted for the effects of orthostasis and basal output.
4. Discussion The aim of this re-analysis was to assess the effect of orthostasis on the reactivity of the SAM system and the HPA axis after psychological stress. We found marked increases in both plasma catecholamines and saliva cortisol as a reaction to a standardized psychosocial stressor (as reported in Nater et al., 2006). In the “standing without stress” condition that was, apart from the stress intervention, identical to the “standing with stress” condition, no substantial increases were found for plasma epinephrine and saliva cortisol, even though subjects were standing for 10 minutes, the same time when a stressor was applied in the “standing with stress” condition. In contrast, an elevation in norepinephrine was found in the “standing without stress” condition, albeit to a lesser degree. Since a considerable part of the variance of endocrine activity was due to orthostasis and basal output alone (74.39% for NE, 17.06% for EP, and 31.09% for cortisol) psychosocial stress-induced changes have therefore to be adjusted downwards, i.e. 25.61%, 82.94%,
Fig. 3. Plasma epinephrine during “standing with stress” and “standing without [w/o] stress” condition. Time of standing is indicated by the black bar. In the “standing with stress” condition, the black bar also indicates the time of the stress test. Error bars indicate SEM. The shaded area illustrates the increase of epinephrine that could be attributed to psychosocial stress adjusted for the effects of orthostasis and basal output.
and 68.91%, change of NE, EP, and cortisol can be attributed to the effect of psychosocial stress. Reactivity of the body to psychological stressors in the laboratory has been widely investigated in order to establish details of the physiological stress response. This effect is usually studied with the subject in a sitting position, although there are a number of paradigms that seek to establish a more realistic setting for the study of stress, and therefore expose the subject to a stressor while being in a standing position. Furthermore, participants are also often required to walk between different rooms while being tested. Physiological stress reactions found in psychological stress paradigms are similar to those found when physiological maneuvers are used to evaluate the function of the body stress systems. It is therefore difficult to separate the relative contribution of “pure” psychological variables (such as uncontrollability and socialevaluative threat) in a psychological stress paradigm in which other circumstances might have an influence, such as subjects being in an upright, standing position or even walking. Using head-up tilt, even the slight increase of around 30° causes a rise in NE, at 60 to 70° not only NE but also EP and cortisol were found to be significantly increased (Hinghofer-Szalkay et al., 1996; Laszlo, Rossler, and Hinghofer-Szalkay, 2001; Sander-Jensen et al., 1986; Vlcek et al., 2008). Indeed, even a change from lying to horizontal prone position results in increased concentrations of both catecholamines (Pump, Talleruphuus, Christensen, Warberg, and Norsk, 2002). Simulating postural changes occurring during the TSST (Mlynarik et al., 2007) resulted in increases of blood pressure, NE, aldosterone and renin activity while ACTH, cortisol and EP remained unchanged. These results show that endocrine variables are very sensitive to bodily posture changes. The adjustments the organism has to make to maintain homeostasis during orthostatic stress constitute simple but distinct stress responses. Rising from a lying to a standing position results in pooling of the blood in the lower extremities after redistribution of blood from the intrathoracic region. A compensatory mechanism controls hypotension by a rise in sympathetic tonus, mediated by an overshoot of NE resulting in an increase in blood pressure (Sever, Osikowska, Birch, and Tunbridge, 1977). Consistent with this, orthostasis for 10 minutes resulted in a significant increase of plasma NE in our study. When psychosocial stress was applied additionally, the NE response was substantially enlarged. In contrast, only small changes in EP were detected due to standing alone (54.66% of the overall EP increase in the “standing with stress” condition due to orthostasis and basal output (see also Nater et al., 2006)). This finding stands in contradiction to the tilt results mentioned above but corresponds to the findings by Mlynarik et al. (2007). There is also evidence that during a well-examined standardized psychosocial stress test, the Trier Social Stress Test (Kirschbaum et al., 1993), postural changes confound autonomic measures with similar heart rate and heart rate variability changes in a control condition asking the participants to assume an upright position for 10 minutes (Rohleder et al., 2006). Replicating and extending this finding, we were able to show an effect of standing during such a control condition on NE while EP seems to be unaffected by postural changes (Nater et al., 2006). This was confirmed by a third study (Mlynarik et al., 2007). Other hormones, e.g. hormones derived from the HPA axis, such as cortisol, may also rise due to orthostatic changes via indirect mechanisms. When a person is in the upright position, fluid leaves the circulation under hydrostatic forces, particularly in the lower limbs. This results in a rise of concentrations of blood components such as red cells, proteins, as well as protein-bound substances (e.g., thyroxine, cortisol, etc.), all of which do not readily pass through the capillary membrane. The temporary hypotension induced by orthostatic stress therefore induces cortisol secretion. In some studies, orthostatic stress resulted in increases of cortisol (Hennig et al., 2000), while in others no changes were found (Hucklebridge et al., 2002). Evidence suggesting that salivary cortisol seems to be unaffected by postural changes in a control condition compared to the TSST (Rohleder et al., 2006) or
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simulating postural changes occurring during the TSST (Mlynarik et al., 2007) is in line with our results. The slight decrease in cortisol during standing in the non-stress condition may reflect falling concentrations due to circadian rhythm (Czeisler and Klerman, 1999). We may therefore assume that the cortisol reactions induced by a standardized psychosocial stress paradigm can be attributed to the psychological characteristics of the stressor alone, which have been suggested to be uncontrollability and social-evaluative threat (Dickerson and Kemeny, 2004b). Hennig et al. (2000) found that upright posture of 20 minutes leads to cortisol increases. However, these findings might be explained by the induction of emotions by the affective pictures the authors used. It has to be mentioned that, in the current study, only two conditions have been included, standing with stress and standing without stress. Further studies might consider a design using four conditions, i.e. including an additional group sitting with and without stress; however, the TSST in its current form does not allow repeated measurement due to habituation effects (Kirschbaum et al., 1995; Schommer, Hellhammer, and Kirschbaum, 2003). Further limitations of our study need to be mentioned. Since we recruited only healthy male young adults, generalization of our results is limited. Future studies will have to examine differences between men and women, as well as to expand the age range of participating subjects. Also, future studies will have to include other dependent variables such as cardiovascular parameters or electrodermal activity (for an examination of mental and postural stress on hemoconcentration and cardiac see also Muldoon et al., 1992; Veldhuijzen Van Zanten et al., 2005). To conclude from our results, standardized psychosocial stressors, such as the TSST, are reliable tools to investigate the mechanisms of the psychological impact stress has on SAM system and HPA axis responses. It is important to note, however, that in addition posture effects may be a significant confounder as orthostasis may determine the magnitude of these physiological responses to psychological demands. Hence, this study should make researchers more cautious when drawing conclusions from a psychosocial stress test alone. Especially rises in NE, as seen in our study, must be attributed in part to orthostatic stress. This is thus the first study to quantify the amount of endocrine changes that can be attributed to this factor in a psychological stress paradigm by comparing a “standing with stress” to a “standing without stress” condition. A possibility to evade this confounding variable in studies solely interested in the effects of psychological stress on the SAM system and the HPA axis may be to expose the subjects in a sitting position during the whole laboratory stress period. If the subject has to be moved from room to room, more innovate solutions, such as moving the subject sitting in a wheelchair, could be applied. However, in ambulatory field studies, as well as in laboratory studies maintaining a certain amount of transferability to real-world situations, the influence of orthostasis and posture changes cannot be excluded completely, and must therefore be taken into account; possibly by creating a nonstress condition simulating postural changes occurring during the stress condition and subtracting the changes induced by orthostasis only.
Financial disclosure All authors reported no biomedical financial interests or potential conflicts of interest.
Acknowledgements This study was supported by a financial grant of the Stiftung für wissenschaftliche Forschung, University of Zurich. U.M.N. and B.D. acknowledge the financial support of the Swiss National Science Foundation. We gratefully acknowledge the help of Roberto La Marca, Ladina Florin, Elvira Abbruzzese, Carole Morandi, and Barbara Gläser in conducting the experiments.
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International Journal of Psychophysiology journal homepage: www.elsevier.com/locate/ijpsycho
Effects of orthostasis on endocrine responses to psychosocial stress Urs M. Nater a,b,⁎, Beate Ditzen b, Jana Strahler a, Ulrike Ehlert b a b
Department of Psychology, Division of Clinical Biopsychology, Philipps University of Marburg, Germany Department of Psychology, Division of Clinical Psychology and Psychotherapy, University of Zürich, Switzerland
a r t i c l e
i n f o
Article history: Received 12 February 2013 Received in revised form 20 August 2013 Accepted 18 October 2013 Available online 28 October 2013 Keywords: HPA axis SAM system Psychosocial stress Catecholamines Cortisol Orthostasis
a b s t r a c t Standardized psychological procedures have been designed to induce physiological stress responses. However, the impact of standing (orthostasis) on the physiological reaction after psychological stress remains unclear. The purpose of the current analysis was to examine and quantify the relative contribution of orthostasis to the physiological stress response by comparing a “standing with stress” to a “standing without stress” condition. We investigated the effect of standing with and without stress on responses of the sympathetic–adrenomedullary (SAM) system and the hypothalamic–pituitary–adrenal (HPA) axis using a standardized psychosocial stress protocol (Trier Social Stress Test) and a non-stress condition in a repeated measures design. Subjects (N = 30) were exposed to both conditions in randomized order and had to maintain a standing, upright position for 10 minutes. In the “standing with stress” condition, significant increases in repeatedly assessed plasma norepinephrine (NE) and epinephrine (EP), as well as in saliva cortisol were found, while in the “standing without stress” condition, no significant changes in plasma epinephrine and saliva cortisol were observed. Calculations of the relative contribution of orthostasis to physiological stress responses revealed that 25.61% of the NE increase, 82.94% of the EP increase, and 68.91% of the cortisol increase, could be attributed to psychosocial stress adjusted for the effects of orthostasis and basal endocrine output. Although these results are indicative for a marked endocrine reaction that is caused by psychosocial stress alone, our findings show that the contribution of orthostasis must be taken into account when interpreting endocrine data collected in a psychosocial stress test. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The role of stress in everyday life and the detrimental consequences it might exert on our body have been widely examined in the past decades (McEwen, 1998). Assessment of hormonal changes in the two main components of the physiological stress system, i.e. the sympathetic-adrenomedullary (SAM) system and the hypothalamic– pituitary–adrenal (HPA) axis, is essential in psychobiological research to examine the effects of stress and a possible relation to future disease risk (Chida and Steptoe, 2010). Reactivity to psychological stressors in the laboratory has been extensively studied. A plethora of psychological stress paradigms has been developed, albeit with mixed results with regard to endocrine reactivity (Biondi and Picardi, 1999). On the other hand, physiological maneuvers and techniques have been used to evaluate the function of the SAM system (e.g., Valsalva maneuver, cold pressor test, static and dynamic exercise, upright posture (Oribe, 1999)) and the HPA axis (e.g., pharmacological stimulation tests (Heim, Ehlert, and Hellhammer, 2000)). ⁎ Corresponding author at: Department of Psychology, Division of Clinical Biopsychology, Philipps University of Marburg, Gutenbergstr. 18, D-35032 Marburg, Germany. Tel.: +49 6421 24080. E-mail addresses: [email protected] (U.M. Nater), [email protected] (B. Ditzen), [email protected] (J. Strahler), [email protected] (U. Ehlert). 0167-8760/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpsycho.2013.10.010
A frequently cited meta-analysis identified two characteristics of acute psychological stressors and tests that reliably induce changes in HPA axis, i.e. uncontrollability and social-evaluative threat (Dickerson and Kemeny, 2004a). A similar analysis for SAM system changes due to psychological stress is not available. However, research suggests that the SAM reacts more broadly and more sensitively to different challenges, such as physical activity or emotional arousal, than does the HPA axis. As can be seen from the above mentioned stressors used to stimulate the SAM system, it is of pivotal importance to have information about how much of the variance is contributed by psychological characteristics (e.g., uncontrollability) and how much of the variance of the effect can be attributed to physical characteristics of the stressor (e.g., standing). In the laboratory, exposure to psychological stressors is usually studied with subjects in a sitting position. However, the simulation of real-life situations, especially when examining the effects of psychosocial stress on human subjects, demands a more realistic setting. Thus, some stress tests have been conducted with the subjects being examined in an upright, standing position (e.g. Kirschbaum, Pirke, and Hellhammer, 1993). Furthermore, subjects are also often required to walk between different rooms in a laboratory (i.e. resting and testing room). Besides these well-established stress tests (Dickerson and Kemeny, 2004a; Gaab et al., 2003; Roy, 2004; Singh, Petrides, Gold, Chrousos, and Deuster, 1999), effects of orthostasis on parameters of the SAM system and the HPA axis have been observed (Bie, Secher,
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Astrup, and Warberg, 1986; Matzen, Secher, Knigge, Bach, and Warberg, 1992; Vlcek, Radikova, Penesova, Kvetnansky, and Imrich, 2008). Assuming the upright posture leads to sharp rises in catecholamine concentrations, especially in norepinephrine (NE). In healthy subjects, changing posture from lying to sitting results in higher concentrations of NE, and in epinephrine (EP) (Cameron et al., 1987), and lying vs. walking increases NE, but not EP concentrations (Robertson et al., 1979). Changing from lying to standing results in higher NE levels (Christensen and Brandsborg, 1976; Cryer, Santiago, and Shah, 1974; Lechin et al., 1995a; Lechin et al., 1995b; Paramore, Fanelli, Shah, and Cryer, 1998; Schofl, Becker, Prank, von zur Muhlen, and Brabant, 1997; Vlcek et al., 2008), whereas in EP, the results are inconclusive: some studies found higher concentrations (Paramore et al., 1998; Schofl et al., 1997; Vlcek et al., 2008), others found no changes (Christensen and Brandsborg, 1976; Cryer et al., 1974; Lechin et al., 1995a; Lechin et al., 1995b). Finally, changing from sitting to standing leads to increased NE and EP (Tulen, Boomsma, and Man in 't Veld, 1999). Orthostatic challenge (via tilt) also results in a rise of other physiological parameters, such as heart period and blood pressure (Hatch, Klatt, Porges, Schroeder-Jasheway, and Supik, 1986). With regard to HPA axis activation, increases in cortisol when changing from a sitting to a standing position have also been found. Plasma cortisol was shown to rise in response to 40 minutes of standing (Abalan et al., 1992). The same was true for salivary cortisol in a balanced cross-over design examining the effect of 20 minutes of sitting, lying, or being in the upright posture with increasing values only in the upright condition (Hennig et al., 2000). However, there are also studies lacking any evidence of postural shift affecting salivary cortisol (Hucklebridge, Mellins, Evans, and Clow, 2002). These findings of postural changes stimulating the SAM system and the HPA axis make it essential to consider orthostasis-induced changes in the respective systems when examining psychosocial stress effects. Studies investigating healthy subjects in either sitting or standing positions or those which require walking from one room to the next, thus risk a possible confounding effect by orthostasis. It might very well be that the observed results are just due to standing or walking rather than due to the psychosocial or mental stress elicited by the task. Addressing the possible confounding effect of orthostasis, Tulen et al. (1999) found no differences in plasma catecholamines between standing and sitting for subjects doing the word–color–Stroop test. There is also evidence that during another well-examined standardized psychosocial stress test, the Trier Social Stress Test (Kirschbaum et al., 1993), postural changes have an impact on autonomic measures. Research indicates that asking the participants to assume an upright position for 10 minutes as a control condition results in heart rate and heart rate variability changes comparable to the TSST condition (Rohleder, Wolf, Maldonado, and Kirschbaum, 2006). In this study, salivary cortisol was unaffected by postural changes during the control condition. Replicating and extending this finding, our group was able to show an effect of standing during such a control condition on NE while EP seemed to be unaffected by postural changes (Nater et al., 2006). These findings were confirmed by a third TSST study that showed increases in heart rate, blood pressure, NE, aldosterone and renin activity to postural changes, while ACTH, cortisol and EP remained unaffected (Mlynarik, Makatsori, Dicko, Hinghofer-Szalkay, and Jezova, 2007). Furthermore, changes of serum cholesterol levels were shown in response to mental stress and standing, but controlling for body shift-induced changes in hemoconcentration diminished the effect of both tasks (Muldoon et al., 1992). The combined effect of postural and mental stress on hemoconcentration was examined in a well-designed study on the effects of head-up-tilt and a mental stressor both alone and in combination on rheological and cardiovascular measures. Changes in blood pressure, total peripheral resistance, plasma volume and hematocrit were highest in the combined condition while cardiac responses did not differ from those to postural stress alone (Veldhuijzen Van Zanten, Thrall, Wasche, Carroll, and Ring, 2005).
In this present work, we will extend previous work by calculating the amount by which psychosocial stress will change endocrine (i.e. catecholamines and cortisol) activity in comparison with changes in body posture. The findings from this study will have implications for the assessment of the relative contribution of orthostasis on psychosocial stress responses and might guide future research in choosing a study design that may take into account orthostasis as a potentially confounding factor. We studied the influence of standing on both catecholamines and cortisol in a balanced exposition to a stress and a no-stress condition in a repeated measures design. This procedure allows us to separate the physiological component and the psychological component in a psychobiological stress paradigm. It is hypothesized that EP and cortisol would not significantly change in response to the “standing without stress” compared to the “standing with stress” condition, while NE is expected to increase in both conditions. It is further expected that a considerable percentage of the change of endocrine markers is just due to postural shift. 2. Methods and materials 2.1. Participants Subjects were recruited through advertisement at the University of Zurich and the Swiss Federal Institute of Technology, Zurich to participate in a larger project on the effects of psychosocial stress on various stress markers. Stress effects on salivary cortisol and α-amylase, plasma catecholamines, and heart rate variability have been previously reported (Nater et al., 2006). Thirty healthy male subjects participated in the study twice, during a “standing with stress” and during a “standing without stress” condition (for subject characteristics see Table 1). All subjects were medication-free and were non-smokers. Subjects with any acute or chronic somatic or psychiatric disorder (as evidenced by self-report) were excluded from the study. Participants were told not to perform any strenuous physical activity 48 hours prior to the experiment and to cease all sporting activities during the time of the study. Intake of ethanol and caffeine was forbidden 18 hours prior to the experiment. At least 60 minutes before the study, subjects were not allowed to eat or brush their teeth so as to avoid gingival bleeding. After the subjects were provided with complete written and oral descriptions of the study, written informed consent was obtained. The subjects were remunerated for participation in the study with 80 Swiss Francs. This study adhered to human experimentation guidelines of the Helsinki Declaration. The study protocol was formally approved by the ethics committee of the University of Zurich and the ethics committee of the Canton of Zurich. 2.2. Procedures All subjects were exposed to a standardized control condition (“standing without stress”) and the Trier Social Stress Test (TSST) (“standing with stress”) with a gap of 2 weeks (±3 days) in between.
Table 1 Subject characteristics and AUCG values. N = 30
“Standing with stress”
“Standing w/o stress”
Age in years Height in cm Weight in kg BMI in kg/m2 AUCG cortisol AUCG epinephrine AUCG norepinephrine
24.8 ± 2.4 182.3 ± 7.7 74.8 ± 9.2 22.5 ± 2.0 518.04 ± 261.68 1 565.17 ± 902.83 12 706.58 ± 4 963.14
306.70 ± 246.86 855.58 ± 542.04 10 116.25 ± 2 840.25
Abbreviations: BMI = body mass index; AUCG = area under the curve with respect to ground (calculated using the trapezoid formula, see Pruessner et al. (2003)).
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Subjects were randomized to either receive the control condition first, or to receive the stress condition first, in order to control for possible sequence effects. The TSST consists of a simulated job interview (5 minutes) followed by a mental arithmetic task (5 minutes) in front of an audience. This task has repeatedly been found to induce profound endocrine changes in 70–80% of the subjects tested (Kirschbaum et al., 1993). In both conditions, an indwelling catheter was inserted into the antecubital vein of the non-dominant arm after arrival. After a baseline of 40 minutes, the experiment started. In both the stress and the control condition, subjects had to change from sitting into the upright, standing position, and remain standing for 10 minutes. Saliva and blood samples were taken 10 minutes before (−10 minutes), immediately before (0 minutes), 5 minutes after beginning (+5 minutes), and directly after the stress test (+15 minutes), with a further sample taken 10 minutes after the stressor (+25 minutes). One additional saliva sample was taken 20 minutes (+35 minutes) after the stressor had ended to capture the delayed reaction of salivary cortisol. All experiments were performed between 2 p.m. and 6 p.m. to control for diurnal variation in endocrine parameters. 2.3. Endocrine assessment Blood samples were taken with EDTA-coated monovettes (Sarstedt, Sevelen/Switzerland), and kept on ice until centrifugation at 3000 rpm for 10 minutes. Six hundred microliters was pipetted in pre-cooled aliquots. Samples were stored at −80 °C until assayed. Plasma norepinephrine and epinephrine were determined by means of HPLC and electrochemical detection after liquid–liquid extraction (as described elsewhere, Ehrenreich et al., 1997). Saliva was collected using Salivette® collection devices (Sarstedt, Sevelen/Switzerland) and stored at −20 °C after completion of the session until biochemical analysis took place. After thawing, saliva samples were centrifuged at 3000 rpm for 5 minutes. Salivary free cortisol was analyzed by using an immunoassay with time-resolved fluorescence detection (Dressendorfer, Kirschbaum, Rohde, Stahl, and Strasburger, 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.
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3. Results The subject characteristics can be found in Table 1. As previously reported, the TSST resulted in significant increases in all hormones (plasma norepinephrine and epinephrine, and salivary cortisol). Comparing those responses to the control condition (“standing without stress”) revealed significant differences between both conditions (for further details please refer to Nater et al., 2006). There were no statistically significant increases in the control condition for EP (Ftime (2.84/85.3) = 1.52; p = 0.22) and salivary cortisol (Ftime (1.39/41.79) = 2.42; p = 0.12), while NE was significantly increased during the control condition (Ftime (2.69/ 80.75) = 39.18; p b 0.001). The areas under the curve with respect to the ground (AUCG) for all parameters were computed (see Table 1). Results show significantly higher concentrations of all parameters during the “standing with stress” condition in comparison to the “standing without stress” condition (AUCG for NE: t29 = 2.98; p = .006; EP: t29 = 5.59; p b .001, cortisol: t28 = 4.66; p b .001). There was no effect of allocation order on AUCG values (NE: F[1,28] = 2.56, p = 0.121; EP: F[1,28] = 0.18, p = 0.677; cortisol: F[1,27] = 0.12, p = 0.737). There was an effect of allocation order on baseline cortisol values on the “standing with stress” day with higher values in those being allocated to this condition first (t28 = 2.37, p = 0.025). No effect was found for baseline values on the “standing without stress” day and for NE and EP values (all p N 0.162). Adding this cortisol baseline value of the stress condition as a covariate, the difference between both conditions was no longer significant (F[1,27] = 0.46, p = 0.502). For all parameters, the relative contribution of standing was analyzed by computing percent of change in the AUCG between the “standing with stress” condition and the “standing without stress” condition on a within-subject basis. The AUCGs in the “standing with stress” condition are 12706.58 pg/ml (NE), 1565.17 pg/ml (EP), and 518.04 nmol/l (cortisol). The change in percentage in the “standing with stress” condition relative to the “standing without stress” condition was 25.61% for NE, 82.94% for EP, and 68.91% for cortisol representing the specific variance of endocrine activity that can be attributed to psychosocial stress adjusted for the effects of orthostasis and basal endocrine output (please see Figs. 1 to 3 for an illustration of these findings).
2.4. Statistical analysis Analyses of variance (ANOVAs) for repeated measures were computed to reveal possible time × condition interactions. All reported results were corrected by the Greenhouse–Geisser procedure where appropriate (violation of sphericity assumption). For catecholamines and cortisol, area under the total response curve with respect to the ground (AUCG) was calculated using the trapezoid formula, according to 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. Due to violation of the normal distribution of some AUCG values for cortisol, norepinephrine, and epinephrine (analyzed by the Kolmogorov–Smirnov test), these measures were log-transformed. Student's t-tests and univariate ANOVAs were computed for comparison of endocrine output between the two conditions. To analyze the relative contribution of psychosocial stress, the percent of change in AUCG between the “standing with stress” condition and the “standing without stress” condition was computed (% change = [AUCG“standing with stress” − AUCG“standing without stress”] / AUCG“standing without stress” * 100) representing the specific variance of total endocrine activity that can be attributed to psychosocial stress. The variance due to changes in body posture and basal activity, taken into account by using AUCG, is thus represented in 100 − % changestress. One further advantage of relating the total output during both conditions is to also control for basal endocrine activity which is included in AUCG. For all analyses, significance level was α = 5%. Unless indicated, all results shown are means ± standard deviation (SD).
Fig. 1. Salivary cortisol during “standing with stress” and “standing without [w/o] stress” condition. Time of standing is indicated by the black bar. In the “standing with stress” condition, the black bar also indicates the time of the stress test. Error bars indicate SEM. The shaded area illustrates the increase of cortisol that could be attributed to psychosocial stress adjusted for the effects of orthostasis and basal output.
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Fig. 2. Plasma norepinephrine during “standing with stress” and “standing without [w/o] stress” condition. Time of standing is indicated by the black bar. In the “standing with stress” condition, the black bar also indicates the time of the stress test. Error bars indicate SEM. The shaded area illustrates the increase of norepinephrine that could be attributed to psychosocial stress adjusted for the effects of orthostasis and basal output.
4. Discussion The aim of this re-analysis was to assess the effect of orthostasis on the reactivity of the SAM system and the HPA axis after psychological stress. We found marked increases in both plasma catecholamines and saliva cortisol as a reaction to a standardized psychosocial stressor (as reported in Nater et al., 2006). In the “standing without stress” condition that was, apart from the stress intervention, identical to the “standing with stress” condition, no substantial increases were found for plasma epinephrine and saliva cortisol, even though subjects were standing for 10 minutes, the same time when a stressor was applied in the “standing with stress” condition. In contrast, an elevation in norepinephrine was found in the “standing without stress” condition, albeit to a lesser degree. Since a considerable part of the variance of endocrine activity was due to orthostasis and basal output alone (74.39% for NE, 17.06% for EP, and 31.09% for cortisol) psychosocial stress-induced changes have therefore to be adjusted downwards, i.e. 25.61%, 82.94%,
Fig. 3. Plasma epinephrine during “standing with stress” and “standing without [w/o] stress” condition. Time of standing is indicated by the black bar. In the “standing with stress” condition, the black bar also indicates the time of the stress test. Error bars indicate SEM. The shaded area illustrates the increase of epinephrine that could be attributed to psychosocial stress adjusted for the effects of orthostasis and basal output.
and 68.91%, change of NE, EP, and cortisol can be attributed to the effect of psychosocial stress. Reactivity of the body to psychological stressors in the laboratory has been widely investigated in order to establish details of the physiological stress response. This effect is usually studied with the subject in a sitting position, although there are a number of paradigms that seek to establish a more realistic setting for the study of stress, and therefore expose the subject to a stressor while being in a standing position. Furthermore, participants are also often required to walk between different rooms while being tested. Physiological stress reactions found in psychological stress paradigms are similar to those found when physiological maneuvers are used to evaluate the function of the body stress systems. It is therefore difficult to separate the relative contribution of “pure” psychological variables (such as uncontrollability and socialevaluative threat) in a psychological stress paradigm in which other circumstances might have an influence, such as subjects being in an upright, standing position or even walking. Using head-up tilt, even the slight increase of around 30° causes a rise in NE, at 60 to 70° not only NE but also EP and cortisol were found to be significantly increased (Hinghofer-Szalkay et al., 1996; Laszlo, Rossler, and Hinghofer-Szalkay, 2001; Sander-Jensen et al., 1986; Vlcek et al., 2008). Indeed, even a change from lying to horizontal prone position results in increased concentrations of both catecholamines (Pump, Talleruphuus, Christensen, Warberg, and Norsk, 2002). Simulating postural changes occurring during the TSST (Mlynarik et al., 2007) resulted in increases of blood pressure, NE, aldosterone and renin activity while ACTH, cortisol and EP remained unchanged. These results show that endocrine variables are very sensitive to bodily posture changes. The adjustments the organism has to make to maintain homeostasis during orthostatic stress constitute simple but distinct stress responses. Rising from a lying to a standing position results in pooling of the blood in the lower extremities after redistribution of blood from the intrathoracic region. A compensatory mechanism controls hypotension by a rise in sympathetic tonus, mediated by an overshoot of NE resulting in an increase in blood pressure (Sever, Osikowska, Birch, and Tunbridge, 1977). Consistent with this, orthostasis for 10 minutes resulted in a significant increase of plasma NE in our study. When psychosocial stress was applied additionally, the NE response was substantially enlarged. In contrast, only small changes in EP were detected due to standing alone (54.66% of the overall EP increase in the “standing with stress” condition due to orthostasis and basal output (see also Nater et al., 2006)). This finding stands in contradiction to the tilt results mentioned above but corresponds to the findings by Mlynarik et al. (2007). There is also evidence that during a well-examined standardized psychosocial stress test, the Trier Social Stress Test (Kirschbaum et al., 1993), postural changes confound autonomic measures with similar heart rate and heart rate variability changes in a control condition asking the participants to assume an upright position for 10 minutes (Rohleder et al., 2006). Replicating and extending this finding, we were able to show an effect of standing during such a control condition on NE while EP seems to be unaffected by postural changes (Nater et al., 2006). This was confirmed by a third study (Mlynarik et al., 2007). Other hormones, e.g. hormones derived from the HPA axis, such as cortisol, may also rise due to orthostatic changes via indirect mechanisms. When a person is in the upright position, fluid leaves the circulation under hydrostatic forces, particularly in the lower limbs. This results in a rise of concentrations of blood components such as red cells, proteins, as well as protein-bound substances (e.g., thyroxine, cortisol, etc.), all of which do not readily pass through the capillary membrane. The temporary hypotension induced by orthostatic stress therefore induces cortisol secretion. In some studies, orthostatic stress resulted in increases of cortisol (Hennig et al., 2000), while in others no changes were found (Hucklebridge et al., 2002). Evidence suggesting that salivary cortisol seems to be unaffected by postural changes in a control condition compared to the TSST (Rohleder et al., 2006) or
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simulating postural changes occurring during the TSST (Mlynarik et al., 2007) is in line with our results. The slight decrease in cortisol during standing in the non-stress condition may reflect falling concentrations due to circadian rhythm (Czeisler and Klerman, 1999). We may therefore assume that the cortisol reactions induced by a standardized psychosocial stress paradigm can be attributed to the psychological characteristics of the stressor alone, which have been suggested to be uncontrollability and social-evaluative threat (Dickerson and Kemeny, 2004b). Hennig et al. (2000) found that upright posture of 20 minutes leads to cortisol increases. However, these findings might be explained by the induction of emotions by the affective pictures the authors used. It has to be mentioned that, in the current study, only two conditions have been included, standing with stress and standing without stress. Further studies might consider a design using four conditions, i.e. including an additional group sitting with and without stress; however, the TSST in its current form does not allow repeated measurement due to habituation effects (Kirschbaum et al., 1995; Schommer, Hellhammer, and Kirschbaum, 2003). Further limitations of our study need to be mentioned. Since we recruited only healthy male young adults, generalization of our results is limited. Future studies will have to examine differences between men and women, as well as to expand the age range of participating subjects. Also, future studies will have to include other dependent variables such as cardiovascular parameters or electrodermal activity (for an examination of mental and postural stress on hemoconcentration and cardiac see also Muldoon et al., 1992; Veldhuijzen Van Zanten et al., 2005). To conclude from our results, standardized psychosocial stressors, such as the TSST, are reliable tools to investigate the mechanisms of the psychological impact stress has on SAM system and HPA axis responses. It is important to note, however, that in addition posture effects may be a significant confounder as orthostasis may determine the magnitude of these physiological responses to psychological demands. Hence, this study should make researchers more cautious when drawing conclusions from a psychosocial stress test alone. Especially rises in NE, as seen in our study, must be attributed in part to orthostatic stress. This is thus the first study to quantify the amount of endocrine changes that can be attributed to this factor in a psychological stress paradigm by comparing a “standing with stress” to a “standing without stress” condition. A possibility to evade this confounding variable in studies solely interested in the effects of psychological stress on the SAM system and the HPA axis may be to expose the subjects in a sitting position during the whole laboratory stress period. If the subject has to be moved from room to room, more innovate solutions, such as moving the subject sitting in a wheelchair, could be applied. However, in ambulatory field studies, as well as in laboratory studies maintaining a certain amount of transferability to real-world situations, the influence of orthostasis and posture changes cannot be excluded completely, and must therefore be taken into account; possibly by creating a nonstress condition simulating postural changes occurring during the stress condition and subtracting the changes induced by orthostasis only.
Financial disclosure All authors reported no biomedical financial interests or potential conflicts of interest.
Acknowledgements This study was supported by a financial grant of the Stiftung für wissenschaftliche Forschung, University of Zurich. U.M.N. and B.D. acknowledge the financial support of the Swiss National Science Foundation. We gratefully acknowledge the help of Roberto La Marca, Ladina Florin, Elvira Abbruzzese, Carole Morandi, and Barbara Gläser in conducting the experiments.
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