Clemens Kirschbaum

Salivary cortisol in psychoneuroendocrine research: Recent developments and applications

The assessment of cortisol in saliva has proven a valid and reliable reflection of the respective unbound hormone in blood. To date, assessment of cortisol in saliva is a widely accepted and frequently employed method in psychoneuroendocrinology. Due to several advantages over blood cortisol analyses (e.g., stress-free sampling, laboratory independence, lower costs) saliva cortisol assessment can be the method of choice in basic research and clinical environments. The determination of cortisol in saliva can facilitate stress studies including newborns and infants and replace blood sampling for diagnostic endocrine tests like the dexamethasone suppression test. The present paper provides an up-to-date overview of recent methodological developments, novel applications as well as a discussion of possible future applications of salivary cortisol determination.

Social support and oxytocin interact to suppress cortisol and subjective responses to psychosocial stress

BACKGROUND The presence of social support has been associated with decreased stress responsiveness. Recent animal studies suggest that the neuropeptide oxytocin is implicated both in prosocial behavior and in the central nervous control of neuroendocrine responses to stress. This study was designed to determine the effects of social support and oxytocin on cortisol, mood, and anxiety responses to psychosocial stress in humans. METHODS In a placebo-controlled, double-blind study, 37 healthy men were exposed to the Trier Social Stress Test. All participants were randomly assigned to receive intranasal oxytocin (24 IU) or placebo 50 min before stress, and either social support from their best friend during the preparation period or no social support. RESULTS Salivary free cortisol levels were suppressed by social support in response to stress. Comparisons of pre- and poststress anxiety levels revealed an anxiolytic effect of oxytocin. More importantly, the combination of oxytocin and social support exhibited the lowest cortisol concentrations as well as increased calmness and decreased anxiety during stress. CONCLUSIONS Oxytocin seems to enhance the buffering effect of social support on stress responsiveness. These results concur with data from animal research suggesting an important role of oxytocin as an underlying biological mechanism for stress-protective effects of positive social interactions.

Salivary cortisol in psychobiological research: an overview.

The measurement of cortisol in saliva provides the basic scientist as well as the clinician with a reliable tool for investigations of hypothalamus-pituitary-adrenal axis activity. Since saliva samples can be obtained stress-free and independent from medically trained personnel this method may be well suited for use in psychobiological studies. This overview intends to give a comprehensive introduction to the method of salivary cortisol assessment and to briefly discuss its application in different scientific disciplines.

Free Cortisol Levels after Awakening: A Reliable Biological Marker for the Assessment of Adrenocortical Activity

In three independent studies, free cortisol levels after morning awakening were repeatedly measured in children, adults and elderly subjects (total n=152). Cortisol was assessed by sampling saliva at 10 or 15 minute intervals for 30-60 minutes, beginning at the time of awakening for two days (Study 1 and 2) or one (Study 3) day, respectively. In all three studies, free cortisol levels increased by 50-75% within the first 30 minutes after awakening in both sexes on all days. Premenopausal women consistently showed a stronger increase with a delayed peak after awakening compared to men on all days. In Study 2, there was a tendency for lower early morning free cortisol levels for women taking oral contraceptives (p=.10). Stability of the area under the curve (AUC) of the early morning free cortisol levels over the three (Study 1 and 2) or two (Study 3) days ranged between r=.39 and r=.67 (p<.001). Neither age, weight, nor smoking showed an effect on baseline or peak cortisol levels. Sleep duration, time of awakening and alcohol consumption also appeared to be unrelated to early morning free cortisol levels. From these data we conclude that in contrast to single assessments at fixed times, early morning cortisol levels can be a reliable biological marker for the individuals adrenocortical activity when measured repeatedly with strict reference to the time of awakening.

Synthesis of a cortisol-biotin conjugate and evaluation as a tracer in an immunoassay for salivary cortisol measurement

Cortisol 3-(o-carboxymethyl)oxime (C3-CMO) and a commercially available biotin-hydrazide derivative were used to synthesize a C3-CMO-biotin conjugate. C3-CMO was converted into a N-hydroxysuccinimide ester derivative which in a second reaction step was allowed to interact with the hydrazide derivative of biotin. This simple-to-perform synthesis yielded a conjugate suitable for use as a tracer in immunoassays for cortisol measurement. Employing biotin as the primary probe in a competitive solid phase immunoassay allows for variable end point determination by means of commercially available labeled avidin or streptavidin derivatives. Streptavidin-Europium was used in conjunction with the DELFIA-system for time-resolved fluorometric end point measurement (TR-FIA) throughout the study. In addition, colorimetric end point determination (ELISA) using streptavidin-alkaline phosphatase as a secondary probe was established and evaluated. Both forms of this non-isotopic assay showed excellent correlation with a commercially available radioimmunoassay adapted for salivary cortisol measurement. The lower detection limit was 0.43 nM for a 50 microliters salivary sample. The intra-assay coefficient of variation was 6.7, 4.7 and 4.0% at cortisol concentrations of 2.2, 5.5 and 13.2 nM, respectively (n = 37), and the corresponding inter-assay coefficients of variation were 9.0, 8.6 and 7.1% (n = 50). The competitive immunoassay requires 1.5 h incubation time and shows robust and reproducible performance. The C3-CMO-biotin conjugate allows for sensitive and flexible end point determination of salivary cortisol levels in immunoassays.

The cortisol awakening response (CAR): facts and future directions.

In humans, the secretion of cortisol from the adrenal glands follows a diurnal cycle with a profound increase after awakening. This increase after awakening, a phenomenon termed the cortisol awakening response (CAR), appears to be a distinct feature of the hypothalamus-pituitary-adrenal (HPA) axis, superimposing the circadian rhythmicity of cortisol secretion. Several studies point towards an important role of the hippocampus and, additionally, other brain structures (e. g. amygdala, prefrontal cortex, suprachiasmatic nucleus) in the regulation of the CAR. There is increasing knowledge that the CAR is influenced by a variety of factors such as gender, health status, and health behavior or stress perception. However, the exact function of the profound cortisol increase after awakening is still not clarified. We hypothesize that the anticipation of the upcoming day is of major relevance for the magnitude of the CAR. The present paper reviews the current knowledge on the neural regulation of the CAR and factors influencing this phenomenon and considerations are addressed concerning the exact function of the CAR.

Burnout, perceived stress, and cortisol responses to awakening.

OBJECTIVE The effects of burnout and perceived stress on early morning free cortisol levels after awakening were investigated in a group of teachers. Previous studies revealed that cortisol levels show a significant increase after awakening, with high intraindividual stability. METHODS Sixty-six teachers from local public schools (42 women and 24 men, mean age 42+/-5 years) were asked to sample saliva for cortisol analysis on 3 consecutive days. On each day, cortisol levels were measured at the time of awakening and 15, 30, and 60 minutes thereafter. On the night before the third day, subjects took 0.5 mg dexamethasone orally for testing glucocorticoid feedback inhibition. Burnout and perceived stress were measured by three different questionnaires. RESULTS Perceived stress correlated with increases of cortisol levels during the first hour after awakening after dexamethasone pretreatment. In addition, teachers scoring high on burnout showed lower overall cortisol secretion on all sampling days, and a higher suppression of cortisol secretion after dexamethasone administration. In the subgroup of teachers with both high levels of perceived stress and high levels of burnout, a lower overall cortisol secretion was observed on the first 2 days, with stronger increases during the first hour after awakening after dexamethasone suppression. This subgroup also showed the lowest self-esteem, the highest external locus of control, and the highest number of somatic complaints. CONCLUSIONS These results demonstrate differential effects of burnout and perceived stress on hypothalamic-pituitary-adrenal axis regulation.

A mechanism converting psychosocial stress into mononuclear cell activation

Little is known about the mechanisms converting psychosocial stress into cellular dysfunction. Various genes, up-regulated in atherosclerosis but also by psychosocial stress, are controlled by the transcription factor nuclear factor κB (NF-κB). Therefore, NF-κB is a good candidate to convert psychosocial stress into cellular activation. Volunteers were subjected to a brief laboratory stress test and NF-κB activity was determined in peripheral blood mononuclear cells (PBMC), as a window into the body and because PBMC play a role in diseases such as atherosclerosis. In 17 of 19 volunteers, NF-κB was rapidly induced during stress exposure, in parallel with elevated levels of catecholamines and cortisol, and returned to basal levels within 60 min. To model this response, mice transgenic for a strictly NF-κB-controlled β-globin transgene were stressed by immobilization. Immobilization resulted in increased β-globin expression, which could be reduced in the presence of the α1-adrenergic inhibitor prazosin. To define the role of adrenergic stimulation in the up-regulation of NF-κB, THP-1 cells were induced with physiological amounts of catecholamines for 10 min. Only noradrenaline resulted in a dose- and time-dependent induction of NF-κB and NF-κB-dependent gene expression, which depended on pertussis-toxin-sensitive G protein-mediated phosphophatidylinositol 3-kinase, Ras/Raf, and mitogen-activated protein kinase activation. Induction was reduced by α1- and β-adrenergic inhibitors. Thus, noradrenaline-dependent adrenergic stimulation results in activation of NF-κB in vitro and in vivo. Activation of NF-κB represents a downstream effector for the neuroendocrine response to stressful psychosocial events and links changes in the activity of the neuroendocrine axis to the cellular response.

HPA axis responses to laboratory psychosocial stress in healthy elderly adults, younger adults, and children: impact of age and gender

Data from five independent studies were reanalyzed in order to investigate the impact of age and gender on HPA axis responses to an acute psychosocial laboratory stress task. The total sample consisted of 102 healthy subjects with 30 older adults (mean age: 67.3 y), 41 young adults (mean age: 23.5 y), and 31 children (mean age: 12.1 y). All participants were exposed to the Trier Social Stress Test (TSST). The stress protocol caused highly significant ACTH and total plasma cortisol responses in older and younger male and female adults (all p<0.0001) as well as salivary free cortisol responses in all six age and gender groups (all p<0.0001). Three-way ANOVAs for repeated measurement were applied to investigate the impact of age and gender on ACTH and cortisol responses. Results showed that the ACTH response to stress was higher in younger adults compared to older adults (main effect: p=0.009, interaction: p=0.06). Post hoc analyses revealed that there was no age effect in the subgroup of women (p=n.s.), while younger men had higher ACTH responses compared to older men (p=0.01). For total plasma cortisol, ANOVA results showed that the pattern of reactivity did not differ between age and gender groups (all interactional effects p=n.s.), although older females had hightened overall cortisol levels compared to the other groups, as proofed in post hoc analyses (all p<0.05). For free salivary cortisol, a significant main effect of gender (p=0.05) and an almost significant three-way-interaction (p=0.09) emerged. Post hoc analyses showed an elevated overall free salivary cortisol response in elderly men compared to elderly women (p=0.006), while no gender differences emerged in neither young adults nor children (both p=n.s.). In sum, the stressor induced significant HPA axis responses in all age and gender groups. The observed ACTH response patterns in young and elderly adults may suggest that a heightened hypothalamic drive in young men decreases with age, resulting in similar ACTH responses in elderly men and women. Alternative interpretations are also discussed. The data also supports the idea of a greater adrenal cortex sensitivity to ACTH signals in young females. Free salivary cortisol responses were elevated in elderly men compared to elderly women, an effect which cannot be explained by gender differences in perceived stress responses to the TSST. It can be speculated if corticosteroid binding globulin (CBG) and/or sex steroids are important modulators of these effects.

Consistent sex differences in cortisol responses to psychological stress.

&NA; In four independent studies, sex differences in cortisol responses to psychological stress were investigated in healthy adolescents and adults (total n = 153). Public speaking and mental arithmetic in front of an audience (Studies 1–3) reliably induced increases in free cortisol levels in both sexes with 2‐ to 4‐fold increases above baseline levels. Mean cortisol responses were 1.5‐ to 2‐fold higher in men compared with women. In Study 3, cortisol profiles were additionally investigated after human corticotropin‐releasing hormone (h‐CRH) and bicycle ergometry until exhaustion. Here, both sexes showed very similar adrenocortical responses. Furthermore, men showed elevated cortisol levels in anticipation of the psychological stress situation without actually having to perform the tasks (Study 4). Under this condition cortisol concentration was unchanged or decreased in women. From these data we conclude that the observed sex difference does not reflect an overall lower responsiveness of the female adrenal cortex. Although these studies do not provide conclusive data, we suggest sex differences in cognitive and/or emotional responses to distressing psychosocial situations which in turn may influence cortisol secretion.