Salivary Cortisol Analysis Research Articles

Cortisol release and heart rate variability in horses during road transport

Based on plasma cortisol concentrations it is widely accepted that transport is stressful to horses. So far, cortisol release during transport has not been evaluated in depth by non-invasive techniques such as analysis of salivary cortisol and faecal cortisol metabolites. Transport also causes changes in heart rate and heart rate variability (HRV). In this study, salivary cortisol, faecal cortisol metabolites, heart rate and HRV in horses transported by road for short (one and 3.5 h) and medium duration (8 h) were determined. With the onset of transport, salivary cortisol increased immediately but highest concentrations were measured towards the end of transport (4.1 ± 1.6, 4.5 ± 2.6, 6.5 ± 1.8 ng/ml in horses transported for one, 3.5 and 8 h, respectively). Faecal cortisol metabolite concentrations did not change during transport, but 1 day after transport for 3.5 and 8 h had increased significantly ( p < 0.01), reflecting intestinal passage time. Compared to salivary cortisol, changes in faecal cortisol metabolites were less pronounced. Heart rate increased and beat-to-beat (RR) interval decreased ( p < 0.05) with the onset of transport. Standard deviation of heart rate increased while root mean square of successive RR differences (RMSSD) decreased in horses transported for 3.5 (from 74 ± 5 to 45 ± 6 ms) and 8 h (from 89.7 ± 7 to 59 ± 7 ms), indicating a reduction in vagal tone. In conclusion, transport of horses over short and medium distances leads to increased cortisol release and changes in heart rate and HRV indicative of stress. The degree of these changes is related to the duration of transport. Salivary cortisol is a sensitive parameter to detect transient changes in cortisol release.

Chronic Infectious Enteritis and Dysregulation of the Hypothalamic Pituitary Adrenal Axis

Purpose: Perturbations of the hypothalamic-pituitary-adrenal axis (HPA) are known to occur in the setting of chronic disease. Cortisol affects many of the body's vital functions. DHEA-S plays an essential role in cortisol dynamics. Concurrent analysis of salivary cortisol and DHEA-S provides an accurate measure of adrenal function. Twenty-four hour circadian panels of salivary cortisol and DHEA-S were obtained in patients with chronic infectious enteritis to investigate adrenal function. Methods: Patients with a diagnosis of chronic infectious enteritis (CIE) who had salivary analysis of cortisol (8AM, 12PM, 4PM, 8PM, 12AM, and 4AM) and DHEA-S (8AM, 8PM, and 12AM) (Sabre Sciences, Inc., Carlsbad, CA) were retrospectively analyzed for circadian adrenal profile compared to normal controls. Results: Overall, 87 patients were analyzed of which there were 37 females and 50 males. The mean age was 44 years. Cortisol levels (Table and Figure 1) showed the average 4AM levels as elevated. DHEA-S levels (Table and Figure 2) showed averages within normal range and an inversion demonstrating an abnormality in the normal circadian rhythm of the HPA.Table 1: Cortisol averages (ng/ml)Table 2: DHEA-S averages (ng/ml)Figure 1Figure 1Conclusion: Patients with CIE develop HPA dysregulation on the basis of salivary cortisol and DHEA-S levels and patterns. This extra intestinal disorder in patients with CIE has broad clinical significance and implications for therapy. Patients with CIE should be examined for adrenal dysfunction with salivary cortisol and DHEA-S measurement as a simple, non-invasive screening test. Patients with this pattern of HPA should be examined for CIE. Disclosure: Dr Borkin-Employee Sabre Sciences, Inc.

Determination of salivary cortisol by liquid chromatography-tandem mass spectrometry

Background: Late evening salivary cortisol concentrations are increasingly used as a screening test in suspected Cushing's syndrome partly because of easy sample collection. The cortisol immunoassays are prone to interference by cross-reacting steroids and therefore there is a need for improvement. The high specificity of an LC-MS assay provides a solution to the problem. Methods: Our liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis utilizes only 0.1 ml of saliva. The samples were extracted with dichloromethane. The extract was evaporated to dryness and cortisol was analysed by LC-MS/MS operating in the negative mode ESI after separation on a reversed-phase column. Results: The calibration curves for analysis of salivary cortisol exhibited consistent linearity and reproducibility in the range of 0.5–20nmol/L. Interassay CVs were 4.3–11% at cortisol concentrations of 0.6–14nmol/L. The lower limit of quantitation (LOQ) was 70pmol/L (signal to noise ratio=10). The mean recovery of the analyte added to saliva samples ranged from 95–106%. The upper limit of the reference range (95%) was 3.0nmol/L. Conclusions: Our method is rapid, sensitive and simple to perform with a routine LC-MS/MS spectrometer.

Liquid chromatography–tandem mass spectrometry analysis of salivary free cortisol: Validation and clinical application

Liquid chromatography–tandem mass spectrometry analysis of salivary free cortisol: Validation and clinical application

Salivary aldosterone to cortisol ratio: Potential screening tool for primary aldosteronism

Increased incidence of primary aldosteronism in the hypertensive population has renewed interest in the role of mineralocorticoids in cardiovascular diseases. Therefore, a simple non-invasive screening tool to detect hypersecretion of aldosterone would be useful. We have previously established and validated a highly sensitive immunoassay for the detection aldosterone in saliva. Given that autonomous hypersecretion of aldosterone should be largely independent from pituitary stimulation, we speculate that the combined analysis of salivary aldosterone (SA) and salivary cortisol (SC) might improve discrimination between normals and Conn's patients (CP). Thus, we analyzed concentrations of SA by our in-house assay and SC by a commercial assay in order to determine the ratio between the two hormones. 14 subjects with confirmed Conn's and 17 healthy volunteers (HV) participated in the study. Daytime saliva samples were collected by Salivettes (Sarstedt, Nümbrecht) between 7am and 11am. Furthermore, in a subgroup (12 CP, 8 HV) an ACTH stimulation test was performed to investigate the influence of pituitary stimulation on the ratio. Un-stimulated SA was 112.7±58.7pg/ml in CP and 65.0±24.4 in HV. The aldosterone to cortisol ratio (ACR) (±SD) was 0.107±0.087 in CP compared to 0.065±0.065 in HV (p<0.001). Post-ACTH stimulation test however, ACR decreased in both groups, 0.014±0.010 and 0.014±0.013 respectively, due to the greatly increased levels of SC compared to the less pronounced increase in SA. This influence of the pituitary ACTH stimulus also can affect the ratio throughout the day. Preliminary data in 8 CP showed that the ACR is higher in the evening compared to daytime due to lower variation in aldosterone values together with decreasing cortisol. 8am values yielded 0.055±0.034 compared to a 0.423±0.565 12pm ACR. So far, our data has shown that at baseline a ratio higher than 0.1 is suggestive of Conn's syndrome. We propose the possibility of using this ratio, elevated in CP compared to controls, as a tool in the diagnosis of primary aldosteronism.

Strategies for salivary cortisol collection and analysis in research with children

Salivary cortisol has emerged in pediatric research as an easy-to-collect, relatively inexpensive, biologic marker of stress. Cortisol is highly variable and is responsive to a wide range of factors that should be considered when incorporating this measure into research with children. Strategies for sample collection include: (1) standardizing the time for sample collection, including baseline samples; (2) using consistent collection materials and methods; (3) controlling for certain drinks, foods, medications, and diagnoses; and (4) establishing procedures and protocols. Other strategies for laboratory analyses include: (1) selecting the appropriate assay and laboratory; (2) identifying units of measure and norms; and (3) establishing quality controls. These strategies control extraneous variables and produce reliable and valid salivary cortisol results.

Behavioral and physiological responses to stabling in naive horses

The purpose of this study was to investigate the response of horses to confinement and isolation in a stable (indoor individual housing) for the first time using behavioral indices, heart rate, and salivary cortisol concentration. Six naive 2-year-old Australian Stock Horse fillies were examined at 4-hour intervals over 24 hours in an outdoor group paddock followed by 24 hours in indoor individual housing. Behavioral observations and scores and heart rates were recorded and saliva samples were taken at each interval. During stabling, all horses became agitated and demonstrated increased vocalization and movement. Behavioral scores were significantly higher in the indoor individual housing ( P < .001). No significant difference in heart rates between the two environments was detected. Mean salivary cortisol did not increase significantly (2 ng/mL ± 1.4 ng/mL in outdoor group paddock vs 2.5 mL ± 1.2 ng/mL in indoor individual housing). No diurnal rhythm in salivary cortisol was evident in either the outdoor group paddock or indoor individual housing. The results of this study highlight that a combination of behavioral and physiological measures allow better understanding of stress, where one measurement may be misleading. First time stabling of horses elicited marked behavioral responses indicative of stress that were not reflected in increased heart rates or salivary cortisol concentrations. The lack of a diurnal cortisol rhythm and the comparatively high basal cortisol concentrations found in the outdoor group paddock environment may imply that the fillies were already stressed; therefore, stabling did not cause further aberrations detectable by salivary cortisol analysis.

Increased responsiveness of the hypothalamus–pituitary–adrenal (HPA) axis to stress in newborns with atopic disposition

In previous studies, atopic patients showed attenuated cortisol responses to psychosocial stress which is suggestive of a hyporeactive hypothalamus–pituitary–adrenal (HPA) axis in this patient group. Regarding the anti-inflammatory role of glucocorticoids, reduced responsiveness of the HPA axis under stress may be one potential explanation of stress-induced exacerbation of atopic symptoms. The present study evaluated whether hyporeactivity of the HPA axis is a feature related to the disposition of atopy rather than a consequence of an ongoing chronic allergic inflammatory process. Newborns with an atopic disposition (parental atopy; n=31) and without atopic disposition (no parental atopy; n=20) were recruited. To further assess atopic disposition, total IgE levels were determined in the cord blood of the neonates. Three days after birth, a blood sample was obtained by a heel prick which is part of a standard pediatric examination. Blood sampling by heel prick is well known to be a significant stressor resulting in activation of the HPA axis in newborns. Analysis of salivary cortisol indicated a significant increase of cortisol levels in the newborns after the stressor with a trend towards an elevated cortisol response in babies with a family history of atopy or with elevated levels of cord IgE (⩾0.5 kU/l). Neonates with a positive parental atopic heritage and elevated cord IgE were found to show significantly elevated cortisol responses to the heel prick stress when compared to newborns without a parental atopic history and normal cord IgE values. Moreover, cord IgE levels were significantly correlated with basal cortisol levels and the cortisol response to the stressor. These findings suggest that atopic disposition in neonates is associated with altered responsiveness of the HPA axis to stress which may increase the vulnerability to develop manifestation of atopy in later life.

Continuity and Change in Emotional Reactivity in Rhesus Monkeys Throughout the Prepubertal Period

We examined continuity and change in emotional reactiviy as assessed by hypothalamic–pituitary–adrenal (HPA) activity in rhesus monkeys. Plasma samples were obtained from mother-peer–reared and surrogate-peer–reared monkeys and assayed for cortisol concentrations at 5 time points in infancy. Both plasma and saliva samples were collected for cortisol assay from the same monkeys as 1-, 2-, and 3-year-old juveniles, respectively. Plasma cortisol concentrations increased during the first 5 months of life and decreased from 1 to 3 years of age. Females consistently had higher plasma cortisol concentrations than males. Plasma cortisol concentrations were lower in surrogate-peer–reared than mother-peer–reared monkeys during the first month of life. Juvenile plasma cortisol concentrations were significantly correlated with infant cortisol levels at all but the earliest time point. Analysis of salivary cortisol from a subset of animals showed a similar age-related decline and the presence of a significant rearing effect, with lower concentrations in surrogate-peer–reared juveniles, but no sex difference. Salivary and plasma cortisol concentrations in these animals were significantly correlated.

Sequence and Seasonal Effects of Salivary Cortisol

Assessments of cortisol levels in saliva have been widely used by both researchers and clinicians as an index of adrenal functioning. Quarterly measurements of morning and evening cortisol levels were determined in a longitudinal study of 147 participants (72 women and 75 men) followed for I year each. The analysis of salivary cortisol revealed no significant gender or age differences in the sample. There was a sequence effect in quarterly cortisol values with a progressive decrease in serial measurements, especially notable in the morning values, as well as a seasonal variation in cortisol levels with significantly higher levels found in winter and fall, compared with spring and summer. The findings in this study suggest that repeated saliva sampling and seasonal variation in cortisol levels may independently affect adrenal response and, therefore, need to be accounted for in longitudinal studies.

Evaluation of hypothalamo-pituitary-adrenal activity in the tree shrew (Tupaia belangeri) via salivary cortisol measurement.

Saliva sampling is frequently used in humans for adrenal glucocorticoid hormone analysis because of advantages such as non-invasiveness, the ease of collection, and storing of the samples. To transfer this advantageous method to laboratory mammals, potentially confounding factors such as stressful handling procedures have to be excluded. In the present study we established a method for collecting saliva for cortisol measurement in freely moving adult male tree shrews (Tupaia belangeri). The practicability of the procedure was demonstrated (i) by stress-induced changes in cortisol levels revealing a significant increase during the stress phase (control = 0.91 nmol/l vs stress = 1.71 nmol/l), and (ii) by reporting no significant differences in salivary cortisol levels before and after performance of a learning task. The present study emphasizes the use of salivary cortisol analysis especially for monitoring acute changes in the hypothalamo-pituitary-adrenal axis activity in male tree shrews.