Anne Helene Garde

Long-term stability of salivary cortisol

The measurement of salivary cortisol provides a simple, non‐invasive, and stress‐free measure frequently used in studies of the hypothalamic‐pituitary‐adrenal axis activity. In research projects, samples are often required to be stored for longer periods of time either because of the protocol of the project or because of lack of funding for analysis. The aim of the present study was to explore the effects of long‐term storage of samples on the amounts of measurable cortisol. Ten pools of saliva were collected on polyester Salivette® tampons from five subjects. After centrifugation the samples were either stored in small vials or spiked to polyester Salivette tampons before analysis for cortisol using Spectria RIA kits. The effects of storage were evaluated by a linear regression model (mixed procedure) on a logarithmic scale. No effects on cortisol concentrations were found after storage of saliva at 5°C for up to 3 months or at −20°C and −80°C for up to one year. In contrast, concentrations of cortisol were found to decrease by 9.2% (95% confidence interval (CI): 3.8%; 14.3%) per month in samples stored at room temperature. Repeated freezing and thawing of samples up to four times before analysis did not affect the measured concentrations of cortisol. The coefficient of residual variation (CVresid) for samples stored on Salivette tampons were twice the CVresid for samples stored in separate vials after centrifugation. In conclusion, centrifuged saliva samples for analysis of cortisol may be stored at 5°C for up to 3 months or at −20°C or −80°C for at least one year. However, long‐term storage at room temperature cannot be recommended. Repeated cycles of freezing and thawing did not appear to affect the concentrations of cortisol.

Sources of biological and methodological variation in salivary cortisol and their impact on measurement among healthy adults : A review

Salivary cortisol is often used in occupational field studies when measuring stress reactions. For purposes of precision and accuracy in measurement, and interpretation of results, it is crucial to know the sources of variability that exert systematic influence on sampling. Variability can be both biological and methodological in origin, and failure to identify its sources may induce erroneous interpretations of Type I and Type II. This review aims to increase our knowledge and provide an overview of the biological and methodological variations of relevance for field measurements of salivary cortisol. It is concluded that: (i) time of sampling has to be carefully registered and included in the statistical analysis; (ii) samples have to be collected at the same time of year in longitudinal designs; (iii) food intake has to be avoided in at least the 2 h before sampling; (iv) vigorous exercise has to be avoided in at least the 2 h, preferably longer, before saliva is collected for measurement of cortisol; (v) variation in results obtained by different laboratory techniques emphasizes use of the same, or otherwise made comparable, laboratory techniques; (vi) concentration of cortisol is dependent on the material of the tampon; (vii) despite the absence of hard evidence, it is recommended that information be collected and results possibly statistically controlled for alcohol consumption, medication, such as oral contraceptives, and treatment for mental diseases; (viii) saliva samples can be stored at −20°C for at least 1 year; (ix) cross‐comparisons of absolute concentrations across studies might be difficult and therefore the establishment of reference intervals for the population studied and method used is recommended.

Evaluation of a radioimmunoassay and establishment of a reference interval for salivary cortisol in healthy subjects in Denmark

A commercial radioimmunoassay (RA) for salivary cortisol was evaluated using certified reference material in water and spiked to pooled saliva in the range 2.1–89.1 nmol/L. A variance component model for describing the effects of age, body mass index (BMI), diurnal variation, gender, days of sick leave during the past year, and smoking habits was established. Reference intervals for salivary cortisol in 120 healthy individuals performing their routine work were established according to the International Union of Pure and Applied Chemistry (IUPAC) and the International Federation of Clinical Chemistry (IFCC). The method evaluation of the certified reference material in water did not show any bias of the method, i.e. recovery was 97% [CI: 94%; 100.9%]. LOD (detection limit) was 1.59 nmol/L. The ratio between analytical and within‐subject variation (CVa/CVi) was 0.14, indicating that the method was adequate for measurement in healthy subjects. Reference intervals were estimated to be from 3.6 to 35.1 nmol/L for samples at the time of awakening (05.27–07.27), 7.6–39.4 nmol/L for peak level in saliva samples collected 20 min after awakening (05.47–07.47), and LOD 10.3 nmol/L for late afternoon samples (17.00–19.00). Reactivity (increase from awakening to 20 min after awakening) was estimated to be 82% [CI: −179; 345%] and recovery (decrease from 20 min after awakening to 18.00) to be 80% [CI: 51; 109%]. Eighteen percent of the subjects showed a decrease in cortisol in saliva from awakening to 20 min after awakening. Salivary cortisol was not affected by age, body mass index, gender, smoking habits or days of sick leave during the past year.

Determination of cortisol in human saliva using liquid chromatography–electrospray tandem mass spectrometry

The aim of this work was to develop a method for determination of cortisol in saliva by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Saliva was sampled on Salivette tubes. These were centrifuged, deuterium-labeled cortisol was added as internal standard and the proteins precipitated by acetonitrile. The supernatant was evaporated, dissolved in methanol acidified with acetic acid and analyzed by LC-MS-MS. The with-in run precision, tested by pooling saliva samples from volunteers and then analyzing these in a single run, was found to be 7% at 0.7 microgram l(-1). The between-run precision was tested by analysis of the same samples at different days and found to be 11% at 2.5 microgram l(-1). The limit of quantification was 0.5 microgram l(-1). The method was applied for analysis of saliva samples from three volunteers during their last week before vacation and the first and second week on vacation. In addition, the method was compared to analysis by an immunological method. The values from the immunological method were 2.7 times higher than the LC-MS-MS results.

Seasonal and biological variation of urinary epinephrine, norepinephrine, and cortisol in healthy women.

BACKGROUND There is a significant circadian and seasonal periodicity in various endocrine functions. The present study describes the within-day and seasonal fluctuation for urinary catecholamines and cortisol and estimates the within- (CV(i)) and between-subject (CV(g)) coefficients of variation for healthy women undertaking their routine work. In addition, index of individuality (I(i)) and power calculations were derived. METHODS Eleven healthy females undertaking their routine life-style at work participated in the study. Each subject collected six samples during 24 h 15 days over a year, giving a total number of 990 samples. Using a random effect analysis of variance, we estimated CV(g) and total within-subject variation (CV(ti)), i.e. combined within-subject and analytical variation, from logarithmically transformed data. Analytical variation was subtracted from CV(ti) to give CV(i). CV(i) was estimated from samples collected monthly during 1 year (CV(iy)), weekly during 1 month (CV(im)), and six to eight times/day (CV(id)). RESULTS A seasonal variation was demonstrated for excretion of epinephrine, norepinephrine, and cortisol standardized with creatinine. Concentrations of urinary epinephrine were higher during June and July compared to the rest of the year, whereas concentrations of urinary cortisol were higher during December and January compared to the rest of the year. Excretion of norepinephrine was lower during working hours and higher during hours off work for June and July compared to the rest of the year. There was a high within- and between-subject variation, which could not be explained by menstrual cycle, behavioral, emotional, or cognitive stress reactions. CONCLUSIONS Despite high biological variation a reasonably low sample size, e.g. 10-50 individuals, is adequate for practical applicability, i.e. studying differences above 150%. The present study recommends to include the sampling time in the statistical evaluation of data and to be aware of the changes in diurnal variations over seasons. When single measurements are to be evaluated, reference intervals are recommended.

Seasonal Variation in Human Salivary Cortisol Concentration

Measurement of cortisol concentration can contribute important information about an individuals ability to adjust to various environmental demands of both physical and psychosocial origin. However, one uncertainty that affects the possibilities of correctly interpreting and designing field studies is the lack of observations of the impact of seasonal changes on cortisol excretion. For this reason, the month‐to‐month changes in diurnal cortisol concentration, the awakening cortisol response (ACR), maximum morning concentration, and fall during the day were studied in a group of 24 healthy men and women 32 to 61 yrs of age engaged in active work. On one workday for 12 consecutive months, participants collected saliva at four time points for determination of cortisol: at awakening, +30 min, +8 h, and at 21:00 h. Data were analyzed by a repeated measures design with month (12 levels) and time‐of‐day (4 levels) as categorical predictors. Cortisol concentrations were analyzed on a log scale. The diurnal pattern of cortisol was similar across months (interaction between month and time of day: p>0.4). The main effects of month and time‐of‐day were statistically significant (p <0.001). Highest concentrations were observed in February, March, and April, and lowest concentrations were observed in July and August. There were no statistically significant effects in any of the other measures, or between men and women. In conclusion, a seasonal variation in salivary cortisol concentrations was detected in an occupationally active population. Thus, seasonal variation needs to be taken into account when designing and evaluating field studies and interventions and when making comparisons across studies.

A Review of the Effect of the Psychosocial Working Environment on Physiological Changes in Blood and Urine

The aim of the present survey was to provide a literary review of current knowledge of the possible association between the psychosocial working environment and relevant physiological parameters measured in blood and urine. Literature databases (PubMed, Toxline, Biosis and Embase) were screened using the key words job, work-related and stress in combination with selected physiological parameters. In total, 51 work place studies investigated the associations between the psychosocial working environment and physiological changes, of which 20 were longitudinal studies and 12 population-based studies. The studied exposures in work place/population-based studies included: job demands (26/8 studies), job control (24/10 studies), social support and/or leadership behaviour (12/3 studies), effort-reward imbalance (three/one studies), occupational changes (four studies), shift work (eight studies), traumatic events (one study) and other (five studies). The physiological responses were catecholamines (adrenaline, noradrenaline) (14 studies), cortisol (28 studies), cholesterol (23 studies), glycated haemoglobinA(1c) (six studies), testosterone (nine studies), oestrogens (three studies), dehydroepiandrosterone (six studies), prolactin (14 studies), melatonin (one study), thyroxin (one study), immunoglobulin (Ig) A (five studies), IgG (four studies), IgM (one study) and fibrinogen (eight studies). In general, fibrinogen and catabolic indicators, defined as energy releasing, were increased, whereas the anabolic indicators defined as constructive building up energy resources were decreased when the psychosocial working environment was perceived as poor. In conclusion, in this review the association between an adverse psychosocial working environment and HbA(1c), testosterone and fibrinogen in serum was found to be a robust and potential candidate for a physiological effect of the psychosocial working environment. Further, urinary catecholamines appear to reflect the effects of shift work and monotonous work.

Diurnal Urinary 6‐Sulfatoxymelatonin Levels among Healthy Danish Nurses during Work and Leisure Time

The present study aims to examine the influence of evening and night shift work, compared to day shift work, on melatonin secretion in nurses in a field setting. Effects were examined during a workday and during a day off. Both fixed schedules and mixed or rotating schedules were studied. In total, 170 nurses were studied: 89 nurses worked fixed schedules, 27 nurses worked the day shift, 12 nurses worked the evening shift, 50 nurses worked the night shift, and 82 nurses worked mixed schedules, with data collected during a day (n=17), evening (n=14), or night shift (n=50). All spot urine samples were collected during 24 h from the participants on a work day and on a day off and were analyzed for 6‐sulphatoxymelatonin. On the day of urine sampling, participants filled in the Karolinska Sleep Diary. Additional information was collected through a telephone interview. Data were analyzed using a mixed procedure with autoregressive covariance structure. The present study showed that shift work affected the concentrations of 6‐sulphatoxymelatonin in the short term by lower excretion in urine from nurses working the night compared to day shift on a workday and on a day off as well. No significant differences were observed between a workday and a day off when doing day and evening shifts, irrespective of mixed and fixed schedules. Sleep length was reduced workdays (from 6.1–6.8 h) among all nurses, compared to days off (from 7.8–8.7 h).

Sleep length and quality, sleepiness and urinary melatonin among healthy Danish nurses with shift work during work and leisure time

BackgroundSleep problems are common effects of shift work. The aim of the present study was to evaluate how different types of shift affect sleep and sleepiness, and to relate sleepiness to urinary 6-sulfatoxymelatonin.MethodsA total of 166 volunteer healthy Danish nurses working day, evening, or night, respectively fixed and mixed schedules were included. Self-reports of sleep were assessed together with real-time sleepiness and spot urine samples analyzed for 6-sulfatoxymelatonin on a workday and a leisure day.ResultsOn a day off the nurses slept longer, with a better quality and reported less sleepiness compared to a workday. Nurses on nightshift reported poorer sleep quality than nurses on other shifts. Sleepiness was highest for nurses on mixed schedules. Concentrations of urinary 6-sulfatoxymelatonin and sleepiness were generally correlated except for nurses working fixed nights.ConclusionsThe poorest sleep quality was observed for nurses in mixed schedules working nights. The lack of correlation between sleepiness and 6-sulfatoxymelatonin on mixed night shift may indicate that the influence of endogenous melatonin is limited.

The effect of the work environment and performance-based self-esteem on cognitive stress symptoms among Danish knowledge workers

Aims: Interpersonal relations at work as well as individual factors seem to play prominent roles in the modern labour market, and arguably also for the change in stress symptoms. The aim was to examine whether exposures in the psychosocial work environment predicted symptoms of cognitive stress in a sample of Danish knowledge workers (i.e. employees working with sign, communication or exchange of knowledge) and whether performance-based self-esteem had a main effect, over and above the work environmental factors. Methods: 349 knowledge workers, selected from a national, representative cohort study, were followed up with two data collections, 12 months apart. We used data on psychosocial work environment factors and cognitive stress symptoms measured with the Copenhagen Psychosocial Questionnaire (COPSOQ), and a measurement of performance-based self-esteem. Effects on cognitive stress symptoms were analyzed with a GLM procedure with and without adjustment for baseline level. Results: Measures at baseline of quantitative demands, role conflicts, lack of role clarity, recognition, predictability, influence and social support from management were positively associated with cognitive stress symptoms 12 months later. After adjustment for baseline level of cognitive stress symptoms, follow-up level was only predicted by lack of predictability. Performance-based self-esteem was prospectively associated with cognitive stress symptoms and had an independent effect above the psychosocial work environment factors on the level of and changes in cognitive stress symptoms. Conclusions: The results suggest that both work environmental and individual characteristics should be taken into account in order to capture sources of stress in modern working life.