Transdisciplinary research in general, and stress research in particular, requires an efficient integration of methodological knowledge of all involved academic disciplines, in order to obtain conclusions of incremental value about the investigated constructs. From a psychologist’s point of view, biochemistry and quantitative neuroendocrinology are of particular importance for the investigation of endocrine stress systems (i.e., the HPA axis, and the SNS). Despite of their fundamental role for the adequate assessment of endocrine activity, both topics are rarely covered by conventional psychological curriculae. Consequently, the transfer of the respective knowledge has to rely on other, less efficient channels of scientific exchange. The present thesis sets out to contribute to this exchange, by highlighting methodological issues that are repeatedly encountered in research on stress-related endocrine activity, and providing solutions to these issues. As outlined within this thesis, modern stress research tends to fall short of an adequate quantification of the kinetics and dynamics of bioactive cortisol. Cortisol has gained considerable popularity during the last decades, as its bioactive fraction is supposed to be reliably determinable from saliva and is therefore the most conveniently obtainable marker of HPA activity. However, a substantial fraction of salivary cortisol is metabolized to its inactivated form cortisone by the enzyme 11β-HSD2 in the parotid glands, which is likely to restrict its utility. Although the commonly used antibody-based quantification methods (i.e. immunoassays) might “involuntarily” qualify this issue to some degree (due to their inherent cross-reactivity with matrix components that are structurally-related to cortisol; e.g., cortisone), they also cause differential within-immunoassay measurement bias: Salivary cortisone has (as compared to salivary cortisol) a substantially longer half-life, which leads to an overestimation of cortisol levels the more time has passed since the onset of the prior HPA secretory episode, and thus tends to distort any inference on the kinetics of bioactive cortisol. Furthermore, absolute cortisol levels also depend on the between-immunoassay variation of antibodies. Consequently, raw signal comparisons between laboratories and studies, which are favorable as compared to effect comparisons, can hardly be performed. This finding also highlights the need for the long-sought standardization of biochemical measurement procedures. The presumably only way to circumvent both issues is to rely on quantification of ultrafiltrated blood cortisol by mass-spectrometric methods. Being partly related to biochemical considerations with research on HPA activity, a second topic arises concerning the operationalization of the construct itself: In contrast to the simple outcome measures like averaged reaction times, inclined stress researchers can only indirectly infer on the sub-processes being involved in HPA activity from longitudinally sampled hormone concentrations. HPA activity can be quantified either by (a) discrete-time, or by (b) continuous-time models. Although the former is the most popular and more convenient approach (as indicated by the overly frequent encounter of ANOVAs and trapezoidal AUC calculations in the field of psychobiological stress research), most discrete time models form rather data-driven, descriptive approaches to quantify HPA activity, that assume the existence of some endocrine resting-state (i.e., a baseline) at the first sampling point and disregard any mechanistic hormonal change occurring in between all following sampling points. Even if one ignores the fact, that such properties are unlikely to pertain to endocrine systems in general, many generic discrete time models fail to account for the specific structure of endocrine data that results from biochemical hormone measurement, as well as from the dynamics of the investigated system. More precisely speaking, cortisol time series violate homoscedasticity, residual normality, and sphericity, which need to be present in order to enable (mixed effects) GLM-based analyses. Neglecting these prerequisites may lead to inference bias unless counter-measures are taken. Such counter-measures usually involve alteration of the scale of hormone concentrations via transformation techniques. As such, a fourth-root transformation of salivary cortisol (being determined by a widely used, commercially available immunoassay) is shown to yield the optimal tradeoff for generating homoscedasticity and residual normality simultaneously. Although the violation of sphericity could be partly accounted for by several correction techniques, many modern software packages for structural equation modeling (e.g., Mplus, OpenMX, Lavaan) also offer the opportunity to easily specify more appropriate moment structures via path notation and therefore to relax the modeling assumptions of GLM approaches to the analysis of longitudinal hormone data. Proceeding from this reasoning, this thesis illustrates how one can additionally incorporate hypotheses about HPA functioning, and thus model all relevant sub-processes that give rise to HPA kinetics and dynamics. The ALT modeling framework being advocated within this thesis, is shown to serve well for this purpose: ALT modeling can recover HPA activity parameters, which are directly interpretable within a physiological framework, that is, distinct growth factors representing the amount of secreted cortisol and velocity of cortisol elimination can serve to interpret HPA reactivity and regulation in a more unambiguous way, as compared to GLM effect measures. For illustration of these advantages on a content level, cortisol elimination after stress induction was found to be elevated as compared to its known pharmacokinetics. While the mechanism behind this effect requires further investigation, its detection would obviously have been more difficult upon application of conventional GLM methods. Further extension of the ALT framework allowed to address a methodological question, which had previously been dealt with by a mere rule of thumb; what’s the optimal threshold criterion, that enables a convenient but comparably accurate classification of individuals whose HPA axis is or is not activated upon encountering a stressful situation? While a rather arbitrarily chosen baseline-to-peak threshold of 2.5 nmol/L was commonly used to identify episodes of secretory HPA activity in time series of salivary cortisol concentrations, a reanalysis of a TSST meta- dataset by means of ALT mixture modeling suggested that this 2.5 nmol/L criterion is overly conservative with modern biochemical measurement tools and should be lowered according to the precision of the utilized assay (i.e., 1.5 nmol/L). In sum, parametric ALT modeling of endocrine activity can provide a convenient alternative to the commonly utilized GLM-based approaches that enables the inference on and quantification of distinct HPA components on a theoretical foundation, and thus to bridge the gap between discrete- and continuous-time modeling frameworks. The implementation of the outlined modeling approaches by the respective statistical syntaxes and practical guidelines being derived from the comparison of cortisol assays mentioned above, are provided in the appendix of the present thesis, which will hopefully help stress researchers to directly quantify the construct they actually intend to assess.

Summary: Salivary alpha-amylase: More than an enzyme - Investigating confounders of stress-induced and basal amylase activity (Dipl.-Psych. Jana Strahler) The hypothalamus-pituitary-adrenal (HPA) axis and the autonomic nervous system (ANS) are two of the major systems playing a role in the adaptation of organisms to developmental changes that threaten homeostasis. The HPA system involves the secretion of glucocorticoids, including cortisol, into the circulatory system. Numerous studies have been published that introduced salivary cortisol to assess HPA axis activity and therefore strengthens its role as an easy obtainable biomarker in stress research that can be monitored easily and frequently. Recent findings suggest a possible surrogate marker of autonomic activity due to autonomic innervation of salivary glands: salivary alpha-amylase (sAA). Up to date, additional methodological research is needed for a better understanding of the advantages and disadvantages of sAA activity in comparison to already established markers of ANS activity. The aim of the present thesis is to further our knowledge of confounders of sAA activity under basal and acute stress conditions and to strengthen the validity of this enzyme as an easy obtainable alternative for ANS testing. After introducing classical and modern stress concepts and stress system physiology (chapter 2), the reader is acquainted with anatomical basics of salivary gland innervation and secretion of salivary proteins, including sAA, due to autonomic innervation (chapter 3 and 4). Afterwards, a more nuanced review of methodological considerations of sAA determination shows gaps of knowledge concerning its usefulness as a marker of ANS activity (chapter 5). Given the fact that the integration of sAA into developmental and aging research is a relative recent phenomenon, several issues have to be addressed before a final conclusion could be drawn. Therefore, we conducted a series of studies incorporating these considerations regarding behavioral correlates of inter- and intraindividual differences in sAA activity with a special emphasis on older adults. Chapter 7 deals with sAA activity under psychological stress conditions in different age groups. Since vulnerability to disease and disease prevalence patterns change with age, it is important to investigate stress reactivity of people in different age groups. We therefore investigated children between 6 and 10 years, because childhood is a sensitive period of growth and development, and thus plays an important role for later life health. Young adults were included to represent the most studied human age group as a reference. Older adults between 59 and 61 years were investigated, because at this age the course is set for the further development of a person’s health in later life, and because autonomic stress responses in older age might be important determinants of cardiovascular and inflammatory aging. Our goal is to test for associations of sAA with more established stress system markers, i.e., salivary cortisol as outcome measurement of HPA reactivity, heart rate (HR) and heart rate variability (HRV) as markers for autonomic reactivity, and to directly compare these responses between different age groups across the life span. Secretion of sAA and cortisol was repeatedly assessed in 62 children, 78 young adults, and 74 older adults after exposure to a standardized psychosocial stressor, the Trier Social Stress Test. In addition, cardiovascular activity was measured in both adult groups. Older adults showed attenuated sAA, HR, and HRV responses. Furthermore, we found higher sAA but lower cortisol at baseline as well as lower sAA and cortisol responses in children. Age by sex interactions were observed only for cortisol with higher responses in older male participants. No associations between the parameters were found. Results in children and young adults confirm previous results. Overall, findings implicate sAA as an alternative or additional autonomic stress marker throughout the life span, with marked and rapid responsiveness to stress in three relevant age groups. The impact of age and chronic stress on basal sAA activity is the center of interest in chapter 8. We therefore assessed diurnal profiles of sAA and salivary cortisol in 27 younger and 31 older competitive ballroom dancers as well as 26 younger and 33 older age- and sex-matched controls. According to the Allostatic Load concept, repeated, non-habituating responses to social-evaluative conditions, which characterize the lives of competitive ballroom dancers, should be associated with stress system dysregulations. Furthermore, we expect to see an increased sympathetic drive associated higher overall alpha-amylase activity in older adults. Analyses revealed an elevated daily overall output of sAA in older adults while there was no effect of age on mean cortisol levels. Alterations of diurnal rhythms were only seen in younger male dancers showing a flattened diurnal profile of sAA and younger dancers and female older dancers showing a blunted diurnal rhythmicity of cortisol. Furthermore, we found a negative correlation between summary indices of basal sAA and the amount of physical activity. In conclusion, higher overall output of sAA in older adults was in line with the phenomenon of a “sympathetic overdrive” with increasing age. Furthermore, a lower output of sAA in people who are more physical active was in line with the hypothesis of an exercise-induced decrease of sympathetic activity. Taken together, results of chapter 7 and 8 show a clear impact of age on sAA activity, either under acute stress or basal conditions. One problem when integrating sAA into developmental and aging research is the use of adrenergic agonists and antagonists what is very common in older adults, i.e. antihypertensive drugs (AD). As well, the previously shown sympathetic overactivity that occurs with normal aging is associated with higher blood pressure (BP). Therefore, chapter 9 deals with a possible impact of high BP and AD on diurnal sAA activity in 79 older adults (33 normotensive adults, 16 medicated vs. 45 hypertensive adults, 34 medicated). Results showed a pronounced rhythm of sAA in all groups. Diurnal profiles differed significantly between men and women with men lacking the typical decrease of sAA in the morning and showing more pronounced alterations throughout the day. An effect of AD on sAA profiles and area under the curve values indicates that subjects not using AD´s show a heightened diurnal profile and a higher total output of sAA. Descriptively, this was also true for hypertensive older adults. Hypertensive subjects and those not using AD showed the highest diurnal output of sAA and the steepest slope. In sum, our results show an impact of antihypertensive medication and a difference between normotensive and hypertensive subjects on characteristics of diurnal sAA activity. Hence, findings are of particular interest in research using sAA as a prognostic indicator of pathological states and processes. Given the fact that hypertension was also shown to be associated with substantial changes of transmitters within the suprachiasmatic nucleus (SCN) - the “biological clock” that receives photic input from retinal glands via the retinohypothalamic pathway - and an altered output from the SCN to the sympathetic nervous system, we broaden the idea of a possible effect of different lighting conditions on morning sAA profiles in chapter 10. In a counterbalanced within-subjects design six men and 16 women of different ages collected sAA morning profiles on two consecutive days with leaving their shutters closed on the one day (= dark) and open their shutters on the other day (= bright). We were able to replicate earlier findings of light-induced changes of salivary cortisol with higher responses during the bright condition. On either day, women showed larger cortisol increases than men. Despite multisynaptic autonomic connections arising from the SCN projecting to multiple organs of the body, we could not find an effect of sunlight on sAA morning profiles. Evidence for circadian clock gene expression in human oral mucosa might account for this result and indicates that peripheral oscillators may act more like integrators of multiple different time cues, e.g. light, food intake, instead of a “master” oscillator (SCN). Results of chapter 7 to 10 provide clear evidence that sAA is heightened in states of autonomic arousal, i.e. stress, aging and hypertension, and that its circadian rhythmicity seems to be regulated rather integrative than directly via efferent input from hypothalamic SCN neurons. In chapter 11 this thesis tries to approach one central question: What is the biological meaning of the findings made? According to this enzyme´s anti-bacterial and digestive action short term changes might not have a biological meaning itself but rather reflect just a small part of multiple coordinated body responses to stressful stimuli. While the sympathetic branch of the ANS mainly stimulates protein secretion, the parasympathetic branch stimulates saliva flow. Acute stress responses might therefore be interpreted as reflecting predominant sympathetic activity together with parasympathetic withdrawal. The same mechanism could also be suitable for the finding of higher diurnal levels of sAA in older adults or hypertensive subjects reflecting a higher peripheral sympathetic tone in these groups. Diurnal profiles of sAA itself may reflect circadian changes in autonomic balance. Circadian rhythms are of great advantage since they enable individuals to anticipate. This pre-adaptation enables the individual to cope with upcoming demands and challenges. Our finding of a relationship between sAA and salivary cortisol what strengthens the relevance of glucocorticoids that were previously shown to be able to phase shift circadian rhythms in cells and tissue. Within a food-related context there is evidence that decreasing levels of sAA in the morning could reflect increases of feeling hungry since sAA systematically increases during food consumption and with the subjective state of satiety. So far, much more research is needed to identify underlying physiological mechanisms of circadian sAA rhythmicity. Taking the next step, future studies will have to focus on the integration of sAA assessment into longitudinal studies and different disease states to prove its applicability as a marker of sympathetic neural functioning in the genesis and prognosis of disease.