Tobias Stalder

Analysis of cortisol in hair – State of the art and future directions

Changes to long-term secretion of the glucocorticoid cortisol are considered to play a crucial role in mediating the link between chronic stress and the development of numerous immune system related diseases. However, obtaining valid assessments of long-term cortisol levels is difficult due to limitations of previous measurement strategies in blood, saliva or urine. This review discusses evidence on a recent methodological development assumed to provide a considerable advancement in this respect: the analysis of cortisol in hair. Being incorporated into the growing hair, hair cortisol concentrations (HCC) are assumed to provide a retrospective reflection of integrated cortisol secretion over periods of several months. Over the past years, supportive evidence has accumulated regarding several fundamental characteristics of HCC, including its validity as an index of long-term systemic cortisol levels, its reliability across repeated assessments and its relative robustness to a range of potential confounding influences. Based on this groundwork, research has now also commenced to utilise HCC for answering more specific questions regarding the role of long-term cortisol secretion in different stress and health-related conditions. The possibility of extending hair analysis to also capture long-term secretion of other steroid hormones (e.g., androgens or estrogens) provides a further intriguing prospect for future research. Given its unique characteristics, the use of hair analysis holds great promise to significantly enhance current understanding on the role of steroid hormones in psychoimmunological research.

Assessment of the cortisol awakening response: Expert consensus guidelines

The cortisol awakening response (CAR), the marked increase in cortisol secretion over the first 30-45 min after morning awakening, has been related to a wide range of psychosocial, physical and mental health parameters, making it a key variable for psychoneuroendocrinological research. The CAR is typically assessed from self-collection of saliva samples within the domestic setting. While this confers ecological validity, it lacks direct researcher oversight which can be problematic as the validity of CAR measurement critically relies on participants closely following a timed sampling schedule, beginning with the moment of awakening. Researchers assessing the CAR thus need to take important steps to maximize and monitor saliva sampling accuracy as well as consider a range of other relevant methodological factors. To promote best practice of future research in this field, the International Society of Psychoneuroendocrinology initiated an expert panel charged with (i) summarizing relevant evidence and collective experience on methodological factors affecting CAR assessment and (ii) formulating clear consensus guidelines for future research. The present report summarizes the results of this undertaking. Consensus guidelines are presented on central aspects of CAR assessment, including objective control of sampling accuracy/adherence, participant instructions, covariate accounting, sampling protocols, quantification strategies as well as reporting and interpreting of CAR data. Meeting these methodological standards in future research will create more powerful research designs, thus yielding more reliable and reproducible results and helping to further advance understanding in this evolving field of research.

Decreased hair cortisol concentrations in generalised anxiety disorder

Previous research examining hypothalamic-pituitary-adrenal (HPA) axis activity in generalised anxiety disorder (GAD) has suggested a general hypercortisolism. These studies have mostly relied on salivary, plasma or urinary assessments, reflecting cortisol secretion over short time periods. The current study utilised the novel method of cortisol assessment in hair to obtain a retrospective index of cortisol secretion over a prolonged period of time. Hair cortisol levels were determined in 15 GAD patients and in 15 age- and gender-matched controls. In addition, participants collected six saliva samples (on awakening, +30 min, 12:00, 16:00, 20:00 h and at bedtime) on two consecutive weekdays for the assessment of the diurnal cortisol profile. Results revealed significantly lower (50-60%) cortisol levels in the first and second 3-cm hair segments of GAD patients compared to those of controls. No significant between-group differences were seen in diurnal cortisol profiles. The hair cortisol findings tentatively suggest that under naturalistic conditions GAD is associated with hypocortisolism. If corroborated by future research, this demonstrates the important qualities of cortisol measurement in hair as an ecologically valid, retrospective index of long-term cortisol secretion and as a marker for psychiatric disorders associated with hypo- or hypercortisolism.

The assessment of cortisol in human hair: Associations with sociodemographic variables and potential confounders

To inform the future use of hair cortisol measurement, we have investigated influences of potential confounding variables (natural hair colour, frequency of hair washes, age, sex, oral contraceptive (OC) use and smoking status) on hair cortisol levels. The main study sample comprised 360 participants (172 women) covering a wide range of ages (1–91 years; mean = 25.95). In addition, to more closely examine influences of natural hair colour and young age on hair cortisol levels, two additional samples comprising 69 participants with natural blond or dark brown hair (hair colour sample) as well as 28 young children and 34 adults (young age sample) were recruited. Results revealed a lack of an effect for natural hair colour, OC use, and smoking status on hair cortisol levels (all ps >0.10). No influence of frequency of hair washes was seen for proximal hair segments (p = 0.335) but for the third hair segment indicating lower cortisol content (p = 0.008). We found elevated hair cortisol levels in young children and older adults (p < 0.001). Finally, men showed higher hair cortisol levels than women (p = 0.002). The present data indicate that hair cortisol measurement provides a useful tool in stress-related psychobiological research when applied with the consideration of possible confounders including age and sex.

Increased cortisol concentrations in hair of severely traumatized Ugandan individuals with PTSD

Previous research has mostly suggested general hypocortisolism in posttraumatic stress disorder (PTSD). However, PTSD is a complex disorder and opposite neuroendocrinological changes have also been reported. Amongst other things, heterogeneous results might be related to differences in sample characteristics as well as methodological factors associated with the assessment of cortisol. The current study used the novel method of hair cortisol analysis to examine cumulative long-term cortisol secretion in a severely traumatized PTSD sample. Hair samples of 10 traumatized individuals with PTSD and 17 traumatized controls without PTSD from a civil war area of Northern Uganda were analyzed. Results revealed that hair samples of PTSD participants contained higher cortisol levels than those of traumatized controls (p<.05). Furthermore, a positive association between hair cortisol levels and the number of lifetime traumatic events was found (p<.05). The current hair cortisol findings suggest that PTSD in severely traumatized individuals who continue to live under stressful conditions might be associated with general hypercortisolism. Future research examining participants after traumatic events at different follow-up periods is needed to determine the specific influence of time interval since traumatization.

Quantitative analysis of steroid hormones in human hair using a column-switching LC-APCI-MS/MS assay

The analysis of steroid hormones in hair is increasingly used in the field of stress-related research to obtain a retrospective index of integrated long-term hormone secretion. Here, most laboratories have so far relied on immunochemical assays originally developed for salivary analyses. Although these assays are fast and easy to perform, they have a reduced reliability and specificity due to cross-reactivity with other substances and are limited to the detection of one hormone at a time. Here, we report the development of a LC-MS/MS-based method for simultaneous identification of endogenous concentrations of seven steroid hormones (cortisol, cortisone, testosterone, progesterone, corticosterone, dehydroepiandrosterone (DHEA) and androstenedione) in human hair. Hair samples were washed with isopropanol and steroid hormones were extracted from 10mg whole, nonpulverized hair by methanol incubation. A column switching strategy for on-line solid phase extraction (SPE) was applied, followed by analyte detection on an AB Sciex API 5000 QTrap mass spectrometer. Results indicated linearity of the method for all steroids over ranges of 0.09-90pg/mg (0.9-900pg/mg for DHEA) with correlation coefficients ranging between 0.9995 and 0.9999. Intra- and inter-assay coefficients of variation were between 3.7 and 9.1%. The limits of quantification (LOQ) were below (or equal to) 0.1pg/mg for all steroids, except of DHEA for which the LOQ was 0.9pg/mg. An analysis of 30 natural hair samples (15 men/15 women) using this method confirmed that all steroid hormones could be quantified at endogenous levels in each individual. In addition, the use of whole hair samples and on-line SPE resulted in a significant reduction in sample throughput times, increasing the applicability of this method for research questions where a larger number of samples needs to be processed.

Hair Cortisol as a Biomarker of Traumatization in Healthy Individuals and Posttraumatic Stress Disorder Patients

BACKGROUND Previous evidence on endocrine correlates of posttraumatic stress disorder (PTSD) has been rather inconsistent. The analysis of cortisol in hair is a recent methodological development that may increase the quality of long-term cortisol assessments in such research. Here, we use this method to closely assess hair cortisol relationships with trauma-related characteristics and PTSD symptom patterns. METHODS Hair cortisol concentrations (HCC), diurnal salivary cortisol, and relevant psychometric data were assessed in matched groups of 28 PTSD patients and 27 traumatized and 32 nontraumatized healthy control subjects. Cortisol levels were quantified by liquid chromatography tandem mass spectrometry. RESULTS Posttraumatic stress disorder patients and traumatized control subjects exhibited 59% and 51% lower HCC than nontraumatized control subjects, respectively. Hair cortisol concentrations were found to be negatively related to the severity of intrusion symptoms, the number of different lifetime traumatic events, the frequency of traumatization, and the time interval since traumatization. The overall pattern of HCC associations was not reflected in short-term salivary cortisol findings. CONCLUSIONS Our results indicate that trauma exposure per se, either in the absence or presence of PTSD, is a crucial correlate of long-term basal cortisol levels. Particularly, the experience of multiple events with a longer time since traumatization and an increased severity of intrusion symptoms may be related to hypocortisolism. The fact that HCC findings were not consistently seen in salivary cortisol data underscores the importance of the method of cortisol assessment and highlights the utility of hair cortisol analyses for future biological psychiatry research.

Classification Criteria for Distinguishing Cortisol Responders From Nonresponders to Psychosocial Stress: Evaluation of Salivary Cortisol Pulse Detection in Panel Designs

Objective Hypothalamic-pituitary-adrenal axis reactivity to acute stimulation is frequently assessed by repeated sampling of salivary cortisol. Researchers often strive to distinguish between individuals who show (responders) and those do not show (nonresponders) cortisol responses. For this, fixed threshold classification criteria, such as a 2.5-nmol/l baseline-to-peak increase, are frequently used. However, the performance of such criteria has not been systematically evaluated. Methods Repeated salivary cortisol data from 504 participants exposed to either the Trier Social Stress Test (TSST; n = 309) or a placebo protocol (n = 195) were used for analyses. To obtain appropriate classifications of cortisol responders versus nonresponders, a physiologically plausible, autoregressive latent trajectory (ALT) mixture model was fitted to these data. Response classifications according to the ALT model and information on the experimental protocol (TSST versus placebo TSST) were then used to evaluate the performance of different proposed classifier proxies by receiver operating characteristics. Results Moment structure of cortisol time series was adequately accounted for by the proposed ALT model. The commonly used 2.5-nmol/l criterion was found to be overly conservative, resulting in a high rate of 16.5% false-negative classifications. Lowering this criterion to 1.5 nmol/l or using a percentage baseline-to-peak increase of 15.5% as a threshold yielded improved performance (39.3% and 26.7% less misclassifications, respectively). Conclusions Alternative classification proxies (1.5 nmol/l or 15.5% increase) are able to effectively distinguish between cortisol responders and nonresponders and should be used in future research, whenever statistical response class allocation is not feasible.

Cortisol in hair and the metabolic syndrome

CONTEXT Although exposure to supraphysiological levels of glucocorticoids is known to contribute to the development of the metabolic syndrome (MetS), the importance of physiological variation in basal cortisol secretion is less clear. This issue can be addressed by using hair cortisol analysis, which for the first time allows the assessment of long-term integrated hormone levels. OBJECTIVE AND DESIGN We used the analysis of cortisol in hair (hairF) to examine associations of long-term cortisol levels with prevalence of MetS and individual cardiometabolic parameters in a large occupational cohort. In additional exploratory analyses, we also studied cardiometabolic associations with hair cortisone levels. PARTICIPANTS Participants included 1258 employees of a large aerospace company (aged 16-64 years; 84.8% males) who partook in a voluntary health assessment. MAIN OUTCOME MEASURES The first 3 cm of scalp-near hair were analyzed for glucocorticoid concentrations using liquid chromatography tandem mass spectrometry. Relevant cardiometabolic risk factors were assessed and MetS was diagnosed (according to 2009 international task force criteria). RESULTS A higher prevalence of MetS was seen in individuals falling into the third (odds ratio 1.71, 95% confidence interval 1.08-2.69) or fourth hairF quartile (odds ratio 2.42, 95% confidence interval 1.55-3.75) compared with the first quartile, in fully adjusted analyses. HairF also showed positive associations with weight-related anthropometric measures (body mass index, waist to hip ratio, waist circumference) and glycated hemoglobin. The exploratory analysis of hair cortisone also indicated relevant associations with cardiometabolic parameters. CONCLUSION Normal physiological differences in long-term cortisol secretion, as assessed in hair, show relevant relationships with MetS and individual cardiometabolic parameters.

Cortisol in hair, body mass index and stress-related measures

Hair cortisol concentrations (HCC) are assumed to reflect integrated cortisol secretion over extended periods of time and may provide a sensitive marker for stress-associated endocrine changes. Here, we report data from two independent studies of 155 (study I) and 58 participants (study II) in which HCC associations with different stress-related measures and body mass index (BMI) were investigated. Consistent evidence for positive associations between HCC and BMI was seen across both studies (study I: r=.33, p<.001; study II: r=.42, p=.001). On the other hand, findings failed to reveal reliable HCC associations with psychosocial variables, showing only a positive relationship with self-reported social overload in study II (r=.29, p=.03) but not with other stress-related measures.