Salivary cortisol for the evaluation of Cushing's syndrome

Cortisol concentrations were measured in matched plasma and salivary samples from 8 healthy controls, 8 patients with Cushing's syndrome and 4 patients suspected of having spontaneous hypercortisolism. In healthy subjects, the circadian rhythm in salivary cortisol paralleled that in plasma. Absence of the diurnal rhythm in Cushing's syndrome was seen in saliva as well as in plasma. After ACTH stimulation, mean peak cortisol in saliva showed a 3-fold increase while in plasma there was a 2.5-fold increment above baseline. Cushing's syndrome, due to pituitary or adrenal adenoma was diagnosed equally well by measuring the cortisol response to cosyntropin in either plasma or saliva. Finally, the low- and high-dose dexamethasone suppression test was reflected equally well in both plasma and saliva. In patients suspected of having Cushing's syndrome dynamic tests can be performed in both plasma and saliva. However, in some samples, the salivary cortisol measurement appears advantageous over plasma cortisol determination.     

Use of saliva for monitoring unbound free cortisol levels in serum

In order to verify the clinical usefulness of saliva in assessing the adrenocortical function, we measured saliva cortisol levels by a radioimmunoassay after extraction with dichloromethane, and compared the results with the levels of serum unbound cortisol determined by the method of equilibrium dialysis. Paired samples of saliva and serum were obtained from 10 healthy male volunteers.

Morning levels of saliva cortisol and serum unbound cortisol were 0.99 ± 0.42 and 1.56 ± 0.54 μg/100 ml, respectively, where serum total cortisol was 19.28 ± 3.56 μg/100 ml. A well-defined diurnal rhythm and a greater response to ACTH stimulation were observed in saliva cortisol than in serum total cortisol. Kinetic study of injected cortisol revealed almost identical values for the compartments of saliva cortisol and serum unbound cortisol. The correlation coefficient (r) between the levels of saliva cortisol and serum unbound cortisol was 0.893 (p < 0.001, n = 150).

From these results we concluded that the measurement of saliva cortisol can be used sufficiently to monitor its unbound free concentrations in serum.     

Direct assay of cortisol in human saliva by solid phase radioimmunoassay and its clinical applications

Direct determination of cortisol (F) in human saliva and its clinical applications were investigated. For this purpose, a solid phase radioimmunoassay (RIA) was carried out. The detection limits for F in 50 and 400 microliter of saliva were 0.125 and 0.0156 micrograms/100 ml, respectively, and the intra-assay coefficient of variation was 5.9--12.2%. Salivary F levels measured by this method were in good agreement with those by the RIA after extraction with dichlormethane reported previously. Salivary F levels were studied in 10 healthy adults with regard to diurnal rhythms, in rapid ACTH tests, and following intravenous injections of 20 mg F, and they showed almost similar changes to serum unbound F levels. Diurnal variations of salivary F in 8 normal children were nearly the same as those in healthy adults. In patients with Cushing's syndrome, neither normal diurnal rhythm of salivary F nor suppressed salivary F with dexamethasone were observed. Patients with adrenocortical insufficiency showed a lack of responsiveness in salivary F levels to stimulation with ACTH or lysin-vasopressin. These findings suggest that salivary F can be measured directly by the solid phase RIA and accurately reflects serum unbound F. The method is simple, accurate and useful for assessing adrenocortical function, especially in pediatric subjects and/or outpatients.     

Predicting the failure of disc surgery by a hypofunctional HPA axis: evidence from a prospective study on patients undergoing disc surgery

Patients with postoperative ongoing sciatic pain have been shown to exhibit reduced cortisol levels along with enhanced IL-6 levels. The aim of the present study was to clarify the relationship between a reduced cortisol secretion and enhanced cytokine levels by performing a prospective study on patients with disc herniation. Twenty-two patients were examined before and after their disc surgery. Twelve healthy, pain-free subjects matched for age, education and gender constituted the control group. The preoperative examinations included the assessment of the diurnal pattern of cortisol secretion and the feedback sensitivity of the hypothalamus-pituitary-adrenal (HPA) axis. Patients' subjective stress levels also were assessed during the preoperative examination. The diurnal pattern of cortisol secretion was again assessed during the postoperative examination. Furthermore, blood samples were collected to measure catecholamine, adrenocorticotropic hormone (ACTH)- and interleukin-6 (IL-6) levels before and after measuring the pressure pain thresholds (PPTs). An assessment of the sensitivity of circulating monocytes to the immunosuppressive effects of glucocorticoids was further included in the postoperative examinations. Failed back syndrome (FBS) patients (n=12) showed a reduced cortisol secretion in the morning hours and enhanced feedback sensitivity of the HPA axis. Furthermore, FBS patients displayed an increased in-vitro production of proinflammatory cytokines and a relative glucocorticoid resistance of pro-inflammatory cytokine producing monocytes as compared to non-FBS patients (n=10) and controls. After PPT measurement FBS patients exhibited an increased norepinephrine but decreased epinephrine response, together with lower ACTH levels and a four times higher plasma IL-6 response. These findings suggest that chronically stressed patients are at a higher risk for a poor surgical outcome as their reduced cortisol secretion promotes the postoperative ongoing synthesis of proinflammatory cytokines.     

Using saliva to measure endogenous cortisol in nursing home residents with advanced dementia

Two research teams determined the feasibility of saliva collection for cortisol measurement in nursing home residents with advanced dementia. Study aims were to: (a) determine if sufficient saliva could be obtained for assay and (b) examine whether cortisol values exhibited range and variability for meaningful interpretation. Useable samples were consistent across sites, suggesting that saliva collection for cortisol assay is a viable method in this setting. Cortisol values showed range and variability. More than half of the residents showed the normal adult pattern of high morning levels decreasing throughout the day. A third of the participants demonstrated an increase in the evening cortisol levels, while the remaining profiles were flat, suggesting hypothalamic-pituitary-adrenal (HPA) dysregulation in this population.     

Time-integrated measurement of corticosteroids in saliva by oral diffusion sink technology.

Saliva, as a medium for assessing adrenocortical function in humans, has many advantages and a few distinct disadvantages. Interpretation of measurements of saliva cortisol is complicated by the contamination of saliva by steroid-binding proteins from blood plasma, enzyme activity in the salivary gland that converts cortisol to cortisone, and the amplification in saliva of the episodic fluctuations in systemic cortisol concentrations. We describe a new measurement technology that rejects artifacts from contamination of saliva by plasma protein, provides for measurement of both cortisol and cortisone, and integrates episodic fluctuations in concentration over a period of hours. This oral diffusion sink technology may greatly enhance the reliable interpretation of corticosteroid concentrations measured in saliva.     

Salivary cortisol and administration of concentrated oral glucose in newborn infants: improved detection limit and smaller sample volumes without glucose interference

Newborn infants are subject to repetitive painful and stressful events during neonatal intensive care. When the baby attempts to cope with a stressful situation the hypothalamus‐pituitary‐adrenal axis is activated, releasing cortisol. The free cortisol response is optimally measured in saliva and saliva samples can be taken easily and without pain. However, saliva is very scarce in infants and saliva stimulants can interfere with analytical methods. Nowadays, sweet solutions are frequently administered to neonates prior to a disturbing procedure in order to reduce pain. The possible interference of sweet solutions with the measurement of salivary cortisol has not yet been documented. The aims of the present study were to further improve the detection limit of the radioimmunoassay used for cortisol analysis and to determine the degree of interference of high concentrations of glucose with the analytical method. By decreasing incubation temperature and prolonging the incubation time it was possible to improve the detection limit of the radio immunoassay (RIA) to 0.5?nmol/L at the same time as the sample volume was decreased to 10?μL saliva. Saliva was collected from full‐term and preterm babies and was sufficient for analysis in 113 out of 116 (97%) samples. Glucose in the concentrations and amounts commonly used for pain relief did not interfere with the RIA method. In conclusion, it is feasible to collect microlitre volumes of saliva and analyse even very low concentrations of cortisol in newborns. It is also possible to offer the baby oral glucose prior to a painful procedure and still reliably measure salivary cortisol.     

Salivary cortisol and administration of concentrated oral glucose in newborn infants: improved detection limit and smaller sample volumes without glucose interference

Newborn infants are subject to repetitive painful and stressful events during neonatal intensive care. When the baby attempts to cope with a stressful situation the hypothalamus-pituitary-adrenal axis is activated, releasing cortisol. The free cortisol response is optimally measured in saliva and saliva samples can be taken easily and without pain. However, saliva is very scarce in infants and saliva stimulants can interfere with analytical methods. Nowadays, sweet solutions are frequently administered to neonates prior to a disturbing procedure in order to reduce pain. The possible interference of sweet solutions with the measurement of salivary cortisol has not yet been documented. The aims of the present study were to further improve the detection limit of the radioimmunoassay used for cortisol analysis and to determine the degree of interference of high concentrations of glucose with the analytical method. By decreasing incubation temperature and prolonging the incubation time it was possible to improve the detection limit of the radio immunoassay (RIA) to 0.5 nmol/L at the same time as the sample volume was decreased to 10 microL saliva. Saliva was collected from full-term and preterm babies and was sufficient for analysis in 113 out of 116 (97%) samples. Glucose in the concentrations and amounts commonly used for pain relief did not interfere with the RIA method. In conclusion, it is feasible to collect microlitre volumes of saliva and analyse even very low concentrations of cortisol in newborns. It is also possible to offer the baby oral glucose prior to a painful procedure and still reliably measure salivary cortisol.     

Aerobic physical activity and salivary cortisol levels among women with a history of breast cancer

BackgroundPhysical activity (PA) helps reduce cancer-related symptoms and improves overall functioning for women with and without a history of breast cancer (BC). Few researchers have examined the associations between PA and physiological stress measures. The aim of this study was to determine whether aerobic PA was associated with diurnal and reactive cortisol patterns, and whether these associations differed for women with and without a history of BC.MethodsParticipants were 25 women with a history of BC and 23 women without a history of BC who self-reported aerobic PA frequency. To assess diurnal cortisol patterns, participants provided five saliva samples collected on two consecutive days at the following times: upon awakening, 30 min after waking, 12 PM, 4 PM, and 9 PM. To measure reactive cortisol patterns, participants provided seven saliva samples collected before, during, and after doing the Trier Social Stress Test.ResultsCortisol patterns differed statistically based on women’s cancer history, whereby women without a history of BC had significantly higher overall cortisol reactivity to an acute stressor, and a marginally significant (p = .05) cancer experience by aerobic PA interaction was observed when analyzing diurnal cortisol data.ConclusionsFindings suggest that PA may not have the same effect on women with and without a history of BC.     

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.     

Rapid saliva processing techniques for near real‐time analysis of salivary steroids and protein

Introduction: Point‐of‐care (POC) measurements using saliva samples have immense potential to assess systemic health and wellbeing, but sample viscosity and contaminants can affect analyses. We sought a portable clean‐up method for whole saliva appropriate for use with POC measurement techniques such as biosensors. Methods: Whole saliva from each of 13 male subjects was split into 5 fractions. Each fraction was treated with a different clean‐up process: a freeze–thaw–centrifuge (FTC) step; centrifugation alone; or passage through a Mini‐UniPrep polyethersulfone filter, cotton Salivette^®, or foam Oracol device. Following clean‐up, each subject's treated saliva fractions were assayed for cortisol, testosterone, dehydroepiandrosterone (DHEA), and proteinconcentrations. The effects of clean‐upmethods on nonspecific binding (NSB) in a biosensor were also assessed. Results: Compared with FTC, no analytes were affected by centrifugation alone. Cotton Salivettes significantly altered all analytes, with increases in cortisol (+64%), testosterone (+126%), and DHEA (off‐scale) levels, and decreased protein (?21%) and biosensor NSB (?75%). Oracol foam devices decreased DHEA levels by 28%. Mini‐UniPrep filtration decreased testosterone (?45%) and DHEA (?66%) concentrations while increasing cortisol (+40%). Conclusion: No method was optimal for all analytes, highlighting the need for validation of saliva treatment methods before their adoption in rapid POC analyses. J. Clin. Lab. Anal. 22:395–402, 2008. © 2008 Wiley‐Liss, Inc.     

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 (CV_a/CV_i) 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.     

Cortisol and wake time in nursing home residents with behavioral symptoms of dementia

Alterations in sleep and behavioral symptoms are consistently reported among nursing home residents with dementia. Disregulation in the hypothalamic-pituitary-adrenal axis (HPA), indexed by basal cortisol levels, offers one explanation. The purpose of this study is to examine the relationship between wake time and cortisol slope in residents with behavioral symptoms. The study included 27 residents aged 71 to 84 years with dementia and behavioral symptoms. Using a within-subject longitudinal design, the researchers documented wake time and collected saliva samples for 4 consecutive days upon waking and at 30 min, 6 hr, and 12 hr after waking. Within-person cortisol slopes were categorized into zero-positive and negative slopes. The zero-positives (35%) exhibited an earlier wake time than the negatives (65%). These preliminary results suggest both a relationship between wake time and HPA diurnal profile and an association between the sleep-wake cycle and cortisol secretion among nursing home residents with dementia.     

Spit: saliva in nursing research, uses and methodological considerations in older adults

Over the last 10 years, interest in the analysis of saliva as a biomarker for a variety of systemic diseases or for potential disease has soared. There are numerous advantages to using saliva as a biological fluid, particularly for nurse researchers working with vulnerable populations, such as frail older adults. Most notably, it is noninvasive and easier to collect than serum or urine. The authors describe their experiences with the use of saliva in research with older adults that examined (a) osmolality as an indicator of hydration status and (b) cortisol and behavioral symptoms of dementia. In particular, the authors discuss the timing of data collection along with data analysis and interpretation. For example, it is not enough to detect levels or rely solely on summary statistics; rather it is critical to characterize any rhythmicity inherent in the parameter of interest. Not accounting for rhythmicity in the analysis and interpretation of data can limit the interpretation of associations, thus impeding advances related to the contribution that an altered rhythm may make to individual vulnerability.     

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.     

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 degrees 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.     

The relative adrenal insufficiency syndrome revisited: which patients will benefit from low-dose steroids?

PURPOSE OF REVIEW: Several clinical studies have given rise to optimism about low-dose steroid treatment in patients with sepsis. It reduces time to shock reversal and may even have a positive effect on mortality. The pathophysiology of the relative adrenal insufficiency syndrome has not yet been determined, and the usefulness of basal and stimulated cortisol levels in diagnosing this syndrome is still uncertain. This review will examine recent evidence to elucidate these questions. RECENT FINDINGS: Studies performed in more than 1000 patients in intensive care show convincingly that in general serum cortisol levels are increased. Basal or stimulated cortisol levels are at best useful to predict mortality in patients in intensive care, not to decide which patients to treat or when to discontinue treatment. Measuring free cortisol concentrations rather than total cortisol concentrations in critically ill patients may lead to new research strategies to identify the mode of action of low-dose steroid treatment. SUMMARY: It has been shown that low-dose corticosteroid administration to catecholamine-dependent patients in septic shock results in shock reversal. There seems to be a relative shortage of cortisol, because low-dose hydrocortisone administration resulting in cortisol levels as much as four times the already increased levels results in shock reversal. Strong evidence for a positive effect on mortality is still lacking, perhaps because of the relatively low number of patients investigated. A very important topic in interpreting studies is that total (free plus protein-bound) cortisol has been measured. Future studies should also measure free cortisol concentrations, which could add to our knowledge of the pathophysiology and treatment of the relative adrenal insufficiency syndrome. On the basis of current knowledge, there is no evidence to support a treatment strategy based on a random or stimulated cortisol level. At the moment, rapid hemodynamic improvement of catecholamine-dependent patients after the administration of low-dose corticosteroids still seems the best available clue to diagnosis.     

Effect of clonidine on plasma cortisol concentrations

There is a debate whether the fall in plasma cortisol concentrations after the early morning dosing of clonidine represents drug effect or normal diurnal variation. We compared the cortisol patterns of children after a 7:30 AM oral dose of clonidine (0.075 or 0.150 mg/m2) with the cortisol concentrations that resulted from diurnal variation. Some subjects had the diurnal study on day 1 and the clonidine study on day 2, whereas for others the testing sequence was reversed. Our results indicate that clonidine, in either dose, does not cause a fall in plasma cortisol levels greater than that with normal diurnal variation.     

Cortisol assays and diagnostic laboratory procedures in human biological fluids

The overview of cortisol physiology, action and pathology is achieved in relation to the hypothalamic-pituitary-adrenal axis alteration by laboratory investigation. The measurements of cortisol and related compound levels in blood, urine and saliva used to study the physiological and pathological cortisol involvement, are critically reviewed. The immunoassay and chromatographic methods for cortisol measurement in the various biological fluids are examined in relation to their analytical performances, reference ranges and diagnostic specificity and sensitivity. Moreover, blood, urine and saliva cortisol level measurements are described taking into account the diagnostic implications. The deduction is that each method requires the definition of its own reference range and its related diagnostic cut-off levels. Thus, this review, stressing the analysis procedures, could help to understand and compare the results of the different assays.