Measurement of late-night salivary cortisol with an automated immunoassay system

Background: Measurement of late-night salivary cortisol concentrations is increasingly used as a screening test in suspected Cushing's syndrome. Cortisol concentrations are typically extremely low in late-night samples and discordant assay-specific reference ranges have been reported. Therefore, the aim of our study was to assess the analytical performance of the first automated cortisol immunoassay specified for salivary measurements and to establish late-night sampling reference-range data for this test. Methods: Salivary cortisol was measured using the Roche Cobas Cortisol assay (Roche Diagnostics). Five salivary pools in different concentration ranges were used to assess the inter-assay imprecision of this test in a two-centre evaluation protocol including two reagent lots. Linearity was tested by serial dilution. Salivary samples were obtained at 23:00 h from 100 apparently healthy volunteers using a commercially available salivary sampling device (Salivette, Sarstedt). A subset of 20 samples was used for method comparison with isotope dilution liquid chromatography-tandem mass spectrometry. Results: Inter-assay coefficients of variation (n=20) between 11.6% and 40.4% were found for mean cortisol concentrations between 12.9 and 2.6 nmol/L, with an estimated functional sensitivity of approximately 5.0 nmol/L. The test also gave linear results in the lowest concentration range between 1.0 and 8.3 nmol/L. Mean late-night salivary cortisol of 5.0 nmol/L was found for healthy individuals; the absolute range was 1.4-16.7 nmol/L, and the 95th percentile was 8.9 nmol/L. Substantially lower concentrations were found with isotope dilution LC-MS/MS compared to immunoassay results (mean concentrations 1.8 and 4.4 nmol/L, respectively). Conclusions: The automated assay investigated was found to offer acceptable analytical performance in the very low concentration range required for late-night salivary cortisol, despite a very short turnaround time. Using this assay, late-night salivary cortisol concentrations below 8.9 nmol/L are typically found in healthy volunteers

Effects of environmental enrichment on behavioral responses to novelty, learning, and memory, and the circadian rhythm in cortisol in growing pigs

Previously we showed that pigs reared in an enriched environment had higher baseline salivary cortisol concentrations during the light period than pigs reared under barren conditions. In the present experiment, it was investigated whether these higher baseline salivary cortisol concentrations were a real difference in cortisol concentration or merely represented a phase difference in circadian rhythm. The effects of different cortisol concentrations on the behavioral responses to novelty and learning and long-term memory in a maze test were also studied in enriched and barren housed pigs. At 9 weeks of age enriched and barren housed pigs did not differ in baseline salivary cortisol concentrations nor in circadian rhythm, but at 22 weeks of age barren housed pigs had a blunted circadian rhythm in salivary cortisol as compared to enriched housed pigs. The differences in baseline salivary cortisol concentrations between enriched- and barren-housed pigs are age-dependent, and become visible after 15 weeks of age. Enriched- and barren-housed piglets did not differ in time spent on exploration in the novel environment test. Barren-housed pigs had an impaired long-term memory in the maze test compared to enriched-housed pigs; however, no differences in learning abilities between enriched- and barren-housed pigs were found. Because blunted circadian cortisol rhythms are often recorded during states of chronic stress in pigs and rats or during depression in humans, it is suggested that the blunted circadian rhythm in cortisol in barren-housed pigs similarily may reflect decreased welfare.

Cortisol and 17-hydroxyprogesterone levels in saliva of healthy neonates - Normative data and relation to body mass index, arterial cord blood pH and time of sampling after birth

The measurement of cortisol and 17-hydroxyprogesterone (17-OHP) in saliva has become a reliable tool for both the scientist and the clinician for studying adrenal cortical function in the adult and the older child. We have now established in parallel normative data for salivary cortisol and 17-OHP levels in healthy neonates. We have asked whether or not there is a circadian rhythm of cortisol and 17-OHP saliva levels in neonates. Furthermore, we have asked whether salivary hormone levels correlated with auxologic and clinical data and time of sampling. Cortisol and 17-OHP levels in saliva samples from 119 healthy neonates (55 girls, 64 boys) were measured using in-house time-resolved fluorescent immunoassays. Saliva samples were obtained using a saliva collecting tube three times a day on the first or second day of life. Gender and gestational age did not influence salivary cortisol and 17-OHP levels. No significant circadian rhythm of salivary hormone levels was detected in this group of newborns. However, body mass index, arterial cord blood pH and time of saliva sampling significantly influenced salivary hormone levels. In conclusion, measurement of cortisol and 17-OHP in saliva is feasible in healthy neonates. The existence of normative data forms the basis for future studies on pathophysiologic states in the newborn period. Copyright (C) 2000 S. Karger AG, Basel

Annexin-A1 protein and its relationship to cortisol in human saliva

Salivary cortisol is commonly used as a clinical biomarker of endocrine status and also as a marker of psychosocial stress. Annexin-A1 (AnxA1) is an anti-inflammatory protein whose expression is modulated by glucocorticoids. Our principal objectives were to (i) detect the presence of and (ii) measure AnxA1 protein in whole human saliva and to (iii) investigate whether salivary cortisol and AnxA1 are correlated in healthy humans. A total of 37 healthy participants (male and female) were used in the study. Saliva was collected using salivette tubes. Salivary cortisol and AnxA1 protein were sampled at between 3 and 6 time points over 24 h and measured for cortisol and AnxA1 protein using specific ELISA's. The presence of salivary AnxA1 protein was confirmed by Western blotting. AnxA1 protein is detectable in whole human saliva, as detected by Western blot analysis and ELISA. A diurnal rhythm was evident in both salivary cortisol (P 0.05), whereas salivary cortisol was significantly elevated between time 0 and 30 min post waking (P < 0.001). AnxA1 protein correlates positively with salivary cortisol, indicating that cortisol is most likely a regulator of AnxA1 in human saliva

Salivary cortisol in the assessment of adrenocortical function in patients with pulmonary tuberculosis

Adrenocortical function was assessed on the basis of changes in salivary cortisol in patients with pulmonary tuberculosis and the findings compared with those in healthy subjects. A method of direct radioimmunoassay of salivary cortisol was standardized and the sensitivity was 0.8 nmol/l. Cortisol levels in saliva were significantly higher in the patients than in the healthy subjects (P < 0.001). The diurnal rhythm of cortisol secretion was disturbed in the patients with a significant increase in salivary cortisol beyond 1800 h. While dexamethasone caused an appreciable suppression (87%), stimulation with ACTH (tetracosactrin) resulted in a marked increase in salivary cortisol, the increase being significantly higher in the healthy subjects than in the patients (P < 0.001). Attempts to classify subjects as positive or negative responders to tetracosactrin based on increases in salivary cortisol in relation to ‘plasma cortisol changes were however not successful, as the agreement between the two methods ranged from 73 to 80 per cent with various criteria used

The Association between Midnight Salivary Cortisol and Metabolic Syndrome in Korean Adults

BackgroundThe common characteristics of metabolic syndrome (MetS) and Cushing's syndrome suggest that excess cortisol may be involved in the pathogenesis of MetS. Salivary cortisol measurements are simple and can be surrogates for plasma free cortisol, which is the most biologically active form. We evaluated the association between levels of midnight salivary cortisol and MetS in Korean adults.MethodsA total of 46 subjects, aged 20 to 70 years, who visited the Health Care Center at Konkuk University Hospital from August 2008 to August 2009 were enrolled. We compared the levels of midnight salivary cortisol in subjects with MetS with those in subjects without MetS. We analyzed the associations between midnight salivary cortisol levels and components of MetS.ResultsMidnight salivary cortisol levels were higher in the MetS group (70±42.4 ng/dL, n=12) than that in the group without MetS (48.1±36.8 ng/dL, n=34) (P=0.001). Positive correlations were observed between midnight salivary cortisol levels and waist circumference, fasting blood glucose, and homeostasis model assessment of insulin resistance. The risk for MetS was significantly higher in subjects with midnight salivary cortisol levels ≥100 ng/dL than in those with levels <50 ng/dL (odds ratio, 5.9; 95% confidence interval, 2.35 to 36.4).ConclusionThe results showed a positive correlation between midnight salivary cortisol levels and MetS, suggesting that hypercortisolism may be related to MetS

Immunity in barren and enriched housed pigs differing in baseline cortisol concentration

It was shown recently that barren housed pigs (small pens, no substrate) have a blunted circadian rhythm of salivary cortisol as compared to enriched housed pigs (large pens with daily fresh bedding). In the light period, enriched housed pigs showed significantly higher concentrations of cortisol in saliva than barren housed pigs, whereas in the dark period, cortisol concentrations were low in both enriched and barren housed pigs. In the present study, the immunological consequences of the difference in baseline salivary cortisol concentration in the light period were evaluated. It appeared that leukocyte and lymphocyte distributions, and in vitro lymphocyte proliferation following ConcanavalineA (ConA) stimulation in the assay using purified lymphocytes were not affected. However, barren and enriched housed pigs did show a different proliferation response to ConA in the whole blood assay. At day 2 of culture, proliferation was higher in barren housed pigs than in enriched housed pigs, whereas at day 4 of culture, proliferation was higher in enriched housed pigs than in barren housed pigs. Lymphocyte proliferation at day 2 of culture in the whole blood assay correlated negatively with plasma cortisol levels, which might thus explain the higher proliferation in barren housed pigs at day 2 of culture. The in vivo humoral and cellular (delayed type hypersensitivity, DTH) immune response to KLH was not affected by housing conditions. We conclude that, although baseline salivary cortisol concentrations differ between enriched and barren housed pigs, immune function appears to be relatively unaffected.

Salivary cortisol determination in patients from the Basque Country with recurrent aphthous stomatitis. A pilot study

Objectives: Stress and anxiety are controversial factors involved in the complex pathogenesis of Recurrent Aph - thous Stomatitis (RAS). The determination of salivary cortisol is a useful, simple and safe test to detect states of high stress or anxiety. The aim of this study is to check for changes in salivary cortisol levels in patients with RAS during periods of active disease. Study design: A measurement of cortisol employing Enzyme-Linked Immuno Sorbent Assay (ELISA) was car - ried out in samples of unstimulated saliva from 20 patients with active lesions of RAS and 10 healthy individuals used as controls. Results: Increased levels of salivary cortisol were detected in 3 cases, all of them within the group of patients with RAS. In none of the control group patients the level of salivary cortisol was increased. The mean level of salivary cortisol was 0.64 mg / dl (range 0.2 to 1.62) for patients with RAS and 0.57 mg / dl (range 0.25 to 1.09) for controls. Conclusion: Salivary cortisol levels are not statistically higher in patients with active lesions of RAS

State anxiety and cortisol reactivity to skydiving in novice versus experienced skydivers

Previous studies have suggested that skydiving, a naturalistic stressor, is associated with increases in self-reported stress, anxiety and cortisol levels. However, it has not been established whether this stress reactivity is altered as a function of repeated exposure to skydiving. This is of interest due to previous observations that cortisol reactivity becomes habituated with repeated exposure to laboratory stressors, however, few studies have investigated such habituation to naturalistic stressors. State anxiety and cortisol reactivity to skydiving were measured in 11 first-time skydivers and 13 experienced skydivers (≥ 30 jumps, mean jumps = 397.6), who were to complete a solo skydive. The novice skydivers reported significantly greater levels of state anxiety prior to the jump; however, there were no differences in pre-jump levels of salivary cortisol. Both groups exhibited significantly elevated salivary cortisol levels immediately post-jump, relative to i) pre-jump and ii) recovery. However, the two groups were indistinguishable with regard to their cortisol reactivity to the skydive. These findings support previous research demonstrating that skydiving elicits acute cortisol activation. Further, they suggest that i) cortisol reactivity does not habituate in experienced jumpers, and ii) that there is lack of concordance between self-reported levels of anxiety and biological stress reactivity in experienced skydivers

Ageing, depression, anxiety, social support and the diurnal rhythm and awakening response of salivary cortisol

The present study compared the cortisol awakening response and diurnal rhythm in 24 young healthy students and 48 community-dwelling older adults. The associations with diurnal cortisol and depression, anxiety and social support were also examined in relation to age. Salivary cortisol was measured over the course of one day: immediately upon awakening, 30 min later, and then 3 h, 6 h, 9 h and 12 h post-awakening. Participants completed a questionnaire measuring symptoms of anxiety and depression and social support was assessed. Older adults exhibited a significantly reduced awakening response, overall cortisol levels, area under the curve (AUC) and diurnal slopes than younger adults, resulting in a flatter diurnal rhythm. Younger adults with higher depression scores had significantly higher overall cortisol and higher levels upon awakening and 30 min post-awakening. In the younger adults, anxiety and depression correlated positively with AUC and the cortisol awakening response (CAR). Older adults with lower social support had a reduced AUC where younger adults with lower social support displayed a larger AUC. These findings suggest that the diurnal rhythm and awakening response of salivary cortisol are significantly reduced in older adults and the associations between anxiety, depression and social support and diurnal cortisol vary with age.\ud \u