Study of temporal variability of salivary cortisol and cortisone by LC-MS/MS using a new atmospheric pressure ionization source.
ABSTRACT: There is a growing interest concerning the relevance of salivary cortisone levels in stress-related research. However, studies investigating morning patterns and day-to-day variability of cortisone versus cortisol levels are lacking. Cortisol and cortisone analysis by liquid chromatography-tandem mass spectroscopy (LC-MS/MS) has been widely used for routine laboratory measurements in the last years. The aim of this study was to develop an ultra-performance LC-MS/MS method for the simultaneous quantification of salivary cortisol and cortisone levels for assessing the temporal variability of these hormones. Saliva samples were collected from 18 healthy volunteers at 0, 15, and 30?min after awakening on each day for 1 week and analysed with the newly developed method. We used a novel atmospheric pressure ionization source, which resulted in high sensitivity and specificity for both cortisol and cortisone as well as higher peak values and signal-to-noise ratio as compared with the electrospray ionization source. Cortisone showed similar morning patterns as cortisol: a 25% and 49% increase in levels at 15 and 30?min after awakening, respectively. Most cortisone indices showed somewhat lower day-to-day variability and were less affected by state-related covariates. We recommend further exploration of the potential of salivary cortisone as a biomarker in stress-related research.
Project description:Context:The assessment absolute bioavailability of oral hydrocortisone is complicated by its saturable binding to cortisol binding globulin (CBG). Previous assessment of bioavailability used a cortisol radioimmunoassay which has cross reactivity with other steroids. Salivary cortisone is a measure of free cortisol and LC-MS/MS is the gold standard method for measuring steroids. We here report the absolute bioavailability of hydrocortisone calculated using serum cortisol and salivary cortisone measured by LC-MS/MS. Methods:14 healthy male dexamethasone suppressed volunteers were administered 20 mg hydrocortisone either intravenously or orally by tablet. Samples of serum and saliva were taken and measured for cortisol and cortisone by LC-MS/MS. Serum cortisol was corrected for saturable binding using published data and pharmacokinetic parameters derived using the program WinNonlin. Results:The mean (95% CI) bioavailability of oral hydrocortisone calculated from serum cortisol, unbound serum cortisol and salivary cortisone was 1.00 (0.89-1.14); 0.88 (0.75-1.05); and 0.93 (0.83-1.05), respectively. Conclusion:The data confirm that, after oral administration, hydrocortisone is completely absorbed. The data derived from serum cortisol corrected for protein binding, and that from salivary cortisone, are similar supporting the concept that salivary cortisone reflects serum free cortisol levels and that salivary cortisone can be used as a non-invasive method for measuring the pharmacokinetics of hydrocortisone.
Project description:Context:Population studies frequently measure cortisol as a marker of stress, and excess cortisol is associated with increased mortality. Cortisol has a circadian rhythm, and frequent blood sampling is impractical to assess cortisol exposure. We investigated measuring salivary cortisone and examined the sampling frequency required to determine cortisol exposure. Methods:Serum and saliva with cortisol and cortisone were measured by liquid chromatography-tandem mass spectrometry in independent cohorts. The relationship between serum cortisol and salivary cortisone was analyzed in cohort 1 using a linear mixed effects model. The resulting fixed effects component was applied to cohort 2. Saliva cannot easily be collected when a patient is sleeping, so we determined the minimum sampling required to estimate cortisol exposure [estimated area under the curve (eAUC)] using 24-hour cortisol profiles (AUC24) and calculated the relative error (RE) for eAUC. Results:More than 90% of variability in salivary cortisone could be accounted for by change in serum cortisol. A single serum cortisol measurement was a poor estimate of AUC24, especially in the morning or last thing at night (RE >68%); however, three equally spaced samples gave a median RE of 0% (interquartile range, -15.6% to 15.1%). In patients with adrenal incidentalomas, eAUC based on three serum cortisol samples showed a difference between those with autonomous cortisol secretion and those without (P = 0.03). Interpretation:Accepting that most people sleep 7 to 8 hours, ?8-hourly salivary cortisone measurements provide a noninvasive method of estimating 24-hour cortisol exposure for population studies.
Project description:Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis and inflammatory systems is a consistent finding in patients with Major Depressive Disorder (MDD). Cortisol is often assessed by measurement of the cortisol awakening response (CAR) and/or diurnal cortisol levels. Some methods of cortisol measurement overestimate cortisol concentration due to detection of other glucocorticoids including the relatively inert cortisone, therefore this study aimed to assess the presence of both cortisol and cortisone, and the cortisol-cortisone catalyzing enzyme 11β-hydroxysteroiddehydrogenase type 1 (11β-HSD1), in depressed patients and controls. Because the HPA axis is known to regulate the body's immune system, relationships between measures of cytokines and cortisol were also assessed. Saliva samples were collected from 57 MDD patients and 40 healthy controls at five post-wakening time points (0, +30, +60, +720 and +750 min). Glucocorticoid concentrations were measured by liquid chromatography mass spectrometry. Whole blood mRNA expression of several inflammatory markers was measured by quantitative polymerase chain reaction. This study replicated the common finding of elevated morning cortisol and reduced CAR reactivity in MDD and found no differences in cortisone or 11β-HSD1 mRNA measures. There was a negative association between interleukin 1-β (IL-1β) mRNA and morning cortisol reactivity within the depressed group, indicating that dysregulation of the HPA axis and immune system may be interconnected.
Project description:OBJECTIVES:We conducted an individual participant meta-analysis to test the hypothesis that cortisol patterns indicative of dysregulated hypothalamic-pituitary-adrenal axis functioning would be prospectively associated with poorer well-being at follow-up. SETTING:Four large UK-based cohort studies. PARTICIPANTS:Those providing valid salivary or serum cortisol samples (n=7515 for morning cortisol; n=1612 for cortisol awakening response) at baseline (age 44-82) and well-being data on the Warwick Edinburgh Mental Wellbeing Scale at follow-up (0-8 years) were included. RESULTS:Well-being was not associated with morning cortisol, diurnal slope or awakening response though a borderline association with evening cortisol was found. Adjusting for sex and follow-up time, each 1 SD increase in evening cortisol was associated with a -0.47 (95% CI -1.00 to 0.05) point lower well-being. This was attenuated by adjustment for body mass index, smoking and socioeconomic position. Between-study heterogeneity was low. CONCLUSIONS:This study does not support the hypothesis that diurnal cortisol is prospectively associated with well-being up to 8 years later. However, replication in prospective studies with cortisol samples over multiple days is required.
Project description:Although an association between both sleep duration and disturbance with salivary cortisol has been suggested, little is known about the long term effects of poor quality sleep on diurnal cortisol rhythm. The aim of this study was to examine the association of poor quality sleep, categorised as recurrent short sleep duration and chronic insomnia symptoms, with the diurnal release of cortisol. We examined this in 3314 participants from an occupational cohort, originally recruited in 1985-1989. Salivary cortisol was measured in 2007-2009 and six saliva samples were collected: (1) waking, (2) waking+0.5h, (3) +2.5h, (4) +8h, (5) +12h and (6) bedtime, for assessment of the cortisol awakening response and the diurnal slope in cortisol secretion. Participants with the first saliva sample collected within 15min of waking and not on steroid medication were examined. Short sleep duration (?5h) and insomnia symptoms (Jenkins scale, highest quartile) were measured in 1997-1999, 2003-2004 and 2007-2009. Recurrent short sleep was associated with a flatter diurnal cortisol pattern. A steeper morning rise in cortisol was observed among those reporting chronic insomnia symptoms at three time points and among those reporting short sleep twice, compared to those who never reported sleep problems. Participants reporting short sleep on three occasions had higher levels of cortisol later in the day, compared to those never reporting short sleep, indicated by a positive interaction with hours since waking (?=0.02 (95% CI: 0.01, 0.03)). We conclude that recurrent sleep problems are associated with adverse salivary cortisol patterns throughout the day.
Project description:Cortisol levels in adults show a sharp decrease from mid-morning to mid-afternoon. Most toddlers take afternoon naps, which is associated with a less mature diurnal pattern characterized by a midday plateau in cortisol secretion. Napping in preschoolers produces a robust cortisol awakening response (CAR), which may account for such maturational differences. This experimental study extends prior work by examining whether the presence and timing of the nap-dependent CAR influences the diurnal cortisol pattern in toddlers.Toddlers (n = 28; 13 females; 30-36 months) followed a strict biphasic sleep schedule (? 12.5h time in bed; ? 90 min nap) for ? 3 days before each of four randomly ordered, in-home cortisol assessments. For each assessment, saliva samples were obtained at morning awakening, ? 09:30, pre-nap, 0, 15, 30, 45, 90, and 135 min post-nap awakening (verified with actigraphy), and ? 19:30. On one day, children napped at their scheduled time, and parents collected saliva samples. On another day, children missed their nap, and parents collected saliva samples at matched times. On two other days, children napped 4h (morning) and 7h (afternoon) after awakening in the morning, during which time researchers collected pre- and post-nap saliva samples. Saliva was assayed for cortisol (?g/dl).Three-level multilevel models were used to estimate the CAR and diurnal cortisol patterns in all four conditions. Compared to the no-nap condition (no observed CAR; b = -0.78, p = 0.65), we found a pronounced cortisol rise following the morning nap (b = 11.00, p < 0.001) and both afternoon naps whether samples were collected by parents (b = 5.19, p < 0.01) or experimenters (b = 4.97, p < 0.01). Napping in the morning resulted in the most robust post-nap cortisol rise (b = 10.21, p < 0.001). Diurnal patterns were analyzed using piecewise growth modeling that estimated linear coefficients for five separate periods throughout the day (corresponding to morning decline, noon decline, post-nap rise, post-nap decline, and evening decline). We observed a significant post-nap rise in cortisol values on the parent-collected afternoon nap (b = 3.41, p < 0.01) and the experimenter-collected morning nap (b = 7.50, p < 0.01) days as compared to the no-nap day (b = -0.17, p = 0.82). No other differences in diurnal profiles were observed between the parent-collected nap and no-nap conditions; however, toddlers had a steeper evening decline on the day of the morning nap compared to the parent-collected afternoon nap (b = 0.30, p < 0.05) and no-nap conditions (b = 0.27, p < 0.05).These well-controlled findings suggest that the presence and timing of daytime naps influence the pattern of diurnal cortisol secretion in toddlers. They also provide support for the hypothesis that napping is the primary state driving the immature midday plateau in cortisol secretion, which becomes more adult-like across childhood. Prior studies of the diurnal cortisol pattern have employed a cubic model, and therefore, have not detected all possible variations due to napping. Our experimental data have important methodological implications for researchers examining associations between the slope of the diurnal cortisol pattern and developmental outcomes, as well as those utilizing afternoon cortisol reactivity protocols in napping children.
Project description:To examine whether day-to-day variations in sleep behaviors, ongoing sleep disturbance, and fatigue predict the cortisol diurnal rhythm in women recently diagnosed as having early-stage breast cancer.Women (N = 130, mean [standard deviation] age = 55.6 [9.4] years) collected saliva 5×/day/2 days for cortisol. Diaries were used to assess prior-day nap duration, nocturnal awakenings, sleep latency, and morning restfulness. Ongoing fatigue and sleep disturbance were measured using the Multidimensional Fatigue Symptom Inventory and the Pittsburg Sleep Quality Inventory. Data were analyzed using a multilevel growth curve modeling.Greater ongoing fatigue (b = 0.035, p = .032), or sleep disturbance (b = 0.026, p = .006) predicted a slower cortisol decline. Greater ongoing fatigue also predicted higher awakening cortisol (b = 0.154, p = .030) and lower cortisol awakening response (CAR; b = -0.146, p = .005). Longer prior-day naps predicted higher CAR (b = 0.042, p = .050) and a steeper cortisol decline (b = -0.035, p = .003). Longer sleep latency predicted both a greater cortisol linear decline (b = -0.013, p < .001) and a greater quadratic slope curvature (b = 0.0007, p < .001). Feeling less rested in the morning predicted lower awakening cortisol (b = -0.187, p = .004), higher CAR (b = 0.124, p = .016), and a slower cortisol decline (b = 0.023, p = .042).Both daily variations in sleep behaviors and ongoing sleep disturbance and fatigue are associated with a disrupted cortisol rhythm. In contrast, prior-day napping is associated with a more robust cortisol rhythm. These findings are particularly relevant to women with breast cancer who often experience sleep disturbance and fatigue. Additional research is needed to determine causal pathways between sleep disturbance and dysregulation of the hypothalamic-pituitary-adrenal axis in patients with breast cancer.
Project description:It is well known that acute stress can lead to a transient increase in cortisol secretion, but the effects of prolonged stress on cortisol secretion are uncertain. This study examines the cross-sectional and longitudinal associations between prolonged perceived stress and salivary cortisol.In 2007, 4467 Danish public service employees participated in a study of stress and mental health, and 3217 participated in a follow-up in 2009. Perceived stress during the past 4 weeks was assessed by Cohen's four item perceived stress scale. Participants were asked to collect saliva 30 min after awakening and at approximately 20:00 in the evening. The cortisol dependence on perceived stress was examined in regression analyses adjusted for effects of potential confounders. We adjusted for a large variation in saliva sampling times by modelling the time trajectory of cortisol concentrations in the morning and in the evening and examined if they were influenced by perceived stress.Perceived stress had no statistically significant effects on the level or time trajectory of morning or evening cortisol, neither cross-sectionally nor longitudinally. The 1 month prevalence of frequently perceived stress was low, approximately 2.5%.Our results did not support the hypothesis that prolonged perceived stress is associated with the level or time trajectory of morning or evening salivary cortisol.
Project description:OBJECTIVES:This study investigated the level of cortisol under different stressful situations and its relationship with sleep and anxiety in female college students. METHODS:Salivary cortisol was measured 6 times a day during a routine period free of examination stress and a stressful period. Area under the curve (AUC) was calculated for cortisol level for awakening response (AUCAG) and during the day (AUCTG). Sleep characteristics and anxiety were measured using an actigraph and the Spielberger State-Trait Anxiety Inventory, respectively, during the different periods. RESULTS:Thirty-six people participated in the study. During the stressful period, anxiety had a positive correlation with sleep efficiency (P = .020), wake after sleep onset (P = .023), and mean wake episodes during sleep (P = .048). Poorer sleep efficiency (P = .014), greater wake after sleep onset (P = .008), and mean wake episodes during sleep (P = .044) were significantly associated with less AUCAG. Trait anxiety was significantly higher in participants with greater AUCTG (P = .008). CONCLUSIONS:Female college students with increased anxiety under the stressful situation slept better. Those with poor sleep showed attenuated morning cortisol secretion. Those with higher trait anxiety had greater cortisol during the day.
Project description:A novel microfluidic chromatography device coupled with tandem mass spectrometry (LC-MS/MS) was utilized for the multiplex analysis of 5 steroids (testosterone, dihydrotestosterone, progesterone, cortisol, cortisone) in human serum. The use of microfluidics allowed for reduction of the chromatographic flow rate to 3?l/min with overall method run times comparable to standard flow LC-MS/MS methods reported in the literature, corresponding to a 150 fold decrease in solvent consumption. Furthermore, a simple sample preparation protocol was employed requiring injection of only 0.5?l of sample, corresponding to a 100-400 fold increase in on-column sensitivity as compared to published standard flow assays. The measured LOQ for both testosterone and progesterone was 0.4ng/mL, representing an improvement over reported literature values obtained by standard flow methods employing comparable sample preparation and large injection volumes. The LOQs for cortisol (1.9ng/mL), cortisone (0.3ng/mL), and dihydrotestosterone (1.4ng/mL) were all within a biologically relevant range. A comparison of clinical serum samples was performed for the analysis of testosterone using this microfluidic LC-MS/MS assay and the Beckman Access II automated antibody-based measurement system. The immunoassay results were systematically higher due to matrix interference which was easily resolved with the increased chromatographic resolution obtained in the microflow LC-MS/MS assay.