Validation of a cortisol enzyme immunoassay and characterization of salivary cortisol circadian rhythm in chimpanzees (Pan troglodytes)

Monitoring concentrations of stress hormones is an important tool for behavioral research and conservation for animals both in the wild and captivity. Glucocorticoids can be measured in mammals as an indicator of stress by analyzing blood, feces, urine, hair, feathers, or saliva. The advantages of using saliva for measuring cortisol concentrations are three-fold: it is minimally invasive, multiple samples can be collected from the same individual in a short timeframe, and cortisol has a relatively short response time in saliva as compared with other materials. The purpose of this study was to: (1) conduct an adrenocorticotropic hormone (ACTH) challenge as a physiological validation for an enzyme immunoassay to measure salivary cortisol in chimpanzees and (2) characterize the circadian rhythm of salivary cortisol in chimpanzees. We determined that salivary cortisol concentrations peaked 45 min following the ACTH challenge, which is similar to humans. Also, salivary cortisol concentrations peaked early in the morning and decreased throughout the day. We recommend that saliva collection may be the most effective method of measuring stress reactivity and has the potential to complement behavioral, cognitive, physiological, and welfare studies.     

Methods of collection for salivary cortisol measurement in dogs

Salivary cortisol has been increasingly used as a measure of stress response in studies of welfare, reaction to stress and human-animal interactions in dogs and other species. While it can be a very useful measure, there are a number of saliva collection issues made evident through studies in the human and animal fields which have not been investigated in the canine species. Collection materials and the volume of saliva that is collected; the use of salivary stimulants; and the effect of food contamination can all dramatically impact cortisol measurement, leading to spurious results. In order to further examine the limitations of the collection method and the effects of collection material and salivary stimulant on salivary cortisol levels, a series of clinical, in vitro and in vivo studies were performed. It was found that there is a large amount of inter- and intra-individual variation in salivary cortisol measurement. Beef flavoring of collection materials leads to unpredictable variability in salivary cortisol concentration. Using salivary stimulants such as citric acid also has the potential to affect cortisol concentration measurement in saliva. Hydrocellulose appears to be a useful collection material for salivary cortisol determination. Recommendations for collection materials and use of salivary stimulants are presented.     

Measurements of Salivary Alpha Amylase and Salivary Cortisol in Hominoid Primates Reveal Within-Species Consistency and Between-Species Differences

Salivary alpha amylase (sAA) is the most abundant enzyme in saliva. Studies in humans found variation in enzymatic activity of sAA across populations that could be linked to the copy number of loci for salivary amylase (AMY1), which was seen as an adaptive response to the intake of dietary starch. In addition to diet dependent variation, differences in sAA activity have been related to social stress. In a previous study, we found evidence for stress-induced variation in sAA activity in the bonobos, a hominoid primate that is closely related to humans. In this study, we explored patterns of variation in sAA activity in bonobos and three other hominoid primates, chimpanzee, gorilla, and orangutan to (a) examine if within-species differences in sAA activity found in bonobos are characteristic for hominoids and (b) assess the extent of variation in sAA activity between different species. The results revealed species-differences in sAA activity with gorillas and orangutans having higher basal sAA activity when compared to Pan. To assess the impact of stress, sAA values were related to cortisol levels measured in the same saliva samples. Gorillas and orangutans had low salivary cortisol concentrations and the highest cortisol concentration was found in samples from male bonobos, the group that also showed the highest sAA activity. Considering published information, the differences in sAA activity correspond with differences in AMY1 copy numbers and match with general features of natural diet. Studies on sAA activity have the potential to complement molecular studies and may contribute to research on feeding ecology and nutrition.     

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.     

Steroid analysis in saliva: an overview

The first report of steroid analysis in saliva was more than thirty years ago. Since that time its popularity has increased due to the attractiveness of non-invasive, repeated and simple stress-free sampling. It has proved a popular sampling fluid for psychobiology, sports medicine, pharmacology and paediatric studies as well as in the area of complementary medicine. In the diagnostic laboratory, salivary progesterone and oestradiol have been used for assessing ovarian function and 17alpha-OH progesterone for the diagnosis of congenital adrenal hyperplasia (CAH). Salivary cortisol is used for investigating adrenal function and recently there has been considerable interest in the use of bedtime salivary cortisol levels as a screening test for Cushing's disease. However, there are several caveats on the use of saliva including collection techniques, the variable matrix of saliva, sensitivity, steroid stability, the presence of binding proteins and reference range anomalies. This brief review will attempt to address these issues and provide a balanced approach to steroid analysis in saliva.     

Association of salivary steroid hormones and their ratios with time-domain heart rate variability indices in healthy individuals

BackgroundStress system consists of the hypothalamicpituitary-adrenal (HPA) axis and the locus caeruleus/norepinephrine-autonomic nervous system (ANS). Traditionally, HPA axis activity is evaluated by measuring its end-product cortisol, while the activity of ANS is assessed using heart rate variability (HRV) indices. Alterations in cortisol levels and HRV measures during laboratory-based stress tasks were extensively studied in previous research. However, scarce data exist on the associations of HRV measures with the levels of other adrenal steroid hormones under baseline conditions. Thus, we aimed to evaluate the activity of the HPA axis by measuring salivary cortisol, cortisone, dehydroepiandrosterone (DHEA) levels, and their ratios and to examine its association with HRV measures in a sample of healthy young and middle-aged adults.MethodsFor each participant (n=40), three data collection sessions taking place at the same time of the day were scheduled within five working days. Participants completed a self-reported questionnaire on sociodemographic and lifestyle characteristics, filled out t h e Perceived Stress Scale and State-Trait Anxiety Inventory. Also, saliva samples were collected, and physiological measures, including resting HR and HRV, were recorded during three data collection sessions.ResultsStatistically significant associations between diminished parasympathetic vagal tone evaluated by time domain HRV measures and higher salivary cortisol, lower DHEA levels, as well as decreased DHEA to cortisol ratio, were found. Also, physiological stress indicators (i.e., HRV) showed greater intraindividual stability compared with biochemical biomarkers (i.e., salivary steroid hormones) within five days.ConclusionsOur findings suggest that both cortisol and DHEA mediate the link between two stress-sensitive homeostatic systems.     

Hand-held monitor of sympathetic nervous system using salivary amylase activity and its validation by driver fatigue assessment

In order to realize a hand-held monitor of the sympathetic nervous system, we fabricated a completely automated analytical system for salivary amylase activity using a dry-chemistry system. This was made possible by the fabrication of a disposable test-strip equipped with built-in collecting and reagent papers and an automatic saliva transfer device. In order to cancel out the effects of variations in environmental temperature and pH of saliva, temperature- and pH-adjusted equations were experimentally determined, and each theoretical value was input into the memory of the hand-held monitor. Within a range of salivary amylase activity between 10 and 140 kU/l, the calibration curve for the hand-held monitor showed a coefficient with R(2)=0.97. Accordingly, it was demonstrated that the hand-held monitor enabled a user to automatically measure the salivary amylase activity with high accuracy with only 30 microl sample of saliva within a minute from collection to completion of the measurement. In order to make individual variations of salivary amylase activity negligible during driver fatigue assessment, a normalized equation was proposed. The normalized salivary amylase activity correlated with the mental and physical fatigue states. Thus, this study demonstrated that an excellent hand-held monitor with an algorithm for normalization of individuals' differences in salivary amylase activity, which could be easily and quickly used for evaluating the activity of the sympathetic nervous system at any time. Furthermore, it is suggested that the salivary amylase activity might be used as a better index for psychological research.     

Nitric oxide modulates salivary amylase and fluid, but not epidermal growth factor secretion in conscious rats

The involvement of the L-arginine/NO pathway in the control of salivary fluid, amylase and epidermal growth factor (EGF) secretion was investigated in conscious rats. For the collection of saliva, an oesophageal cannula was implanted. To obtain steady secretion, submaximal carbachol background infusion was given. Different treatments included NO synthase inhibitor N(G)-nitro-L-arginine (NOLA; with or without phentolamine, propranolol), L-arginine, D-arginine and NO donor 3-morpholinosydnonimine (SIN-1) administration. Volume, amylase activity and EGF output in the secreted fluid were determined in 30 min mixed saliva samples. Carbachol infusion alone produced a modest, sustained salivary fluid and amylase secretion. NOLA (30 mg/kg) further increased both fluid (p<0.001) and amylase outputs (p<0.001). These latter effects were prevented by L-arginine but not by D-arginine or by phentolamine. Propranolol administration decreased both fluid and amylase secretion below the carbachol plateau, and NOLA did not modify this suppressed secretory rate. SIN-1 did not alter either volume or amylase secretion. Interestingly, NOLA given without carbachol did not modify salivation. Neither carbachol nor NOLA changed salivary EGF output. The present results suggest that the L-arginine/NO pathway has a modulatory role in the cholinergic control of salivary amylase secretion, but not in EGF output. The mechanisms of inhibitory action of NO on salivary fluid and amylase secretion remain to be identified.     

Level of alpha amylase activity in human saliva as a non-invasive biochemical marker of sleep deprivation

To investigate the usefulness of the enzyme salivary alpha amylase as a biochemical marker of sleep deprivation in human subjects. Total 168 healthy school-going adolescents studying in 9th grade were selected randomly from morning shift (n = 84) and dayshift (n = 84) schools. The study was undertaken longitudinally for a period of 2 years. Study encompassed administration of questionnaire and collection of saliva samples from the participants. Activity of salivary alpha amylase (sAA) activity was estimated spectrophotometrically and statistical analysis was performed to determine the association between sAA activity and sleep duration. Excessive daytime sleepiness among students was also studied in association with sAA activity. sAA activity of students was found to have a negative correlation with the duration of sleep and a positive correlation with their level of sleepiness. Morning shift students were found to have significantly less sleep and correspondingly higher sAA activity as compared to dayshift students. A significant increase in the sAA activity was noticed in the second year as the students progressed from 9th to 10th grade. Higher amylase activity was also observed in sleep deprived students suffering from excessive daytime sleepiness irrespective of school timings. Salivary alpha amylase activity increases in saliva in response to sleep deprivation. School timings may modulate sleep duration of students. Present finding reveals that sAA could be an appropriate non-invasive biochemical marker for the objective assessment of sleep deprivation among individuals as well as at population level.

     

Techniques for collecting saliva from awake, unrestrained, adult monkeys for cortisol assay

Cortisol levels serve as an index of pituitary-adrenal activity in nonhuman primates. In adult monkeys, cortisol is normally measured in blood (typically requiring restraint or sedation) or urine (reflecting a state rather than point estimate). In contrast, saliva collection is less invasive than drawing blood and allows for repeated sampling within a short period of time. Although protocols exist for collecting saliva from young monkeys, these procedures are inadequate for awake, unrestrained adult animals. Our laboratory has developed two methods for collecting saliva from adult rhesus monkeys: a "screen" method, which involves licking screen-covered gauze, and a "pole" method, which involves sucking and chewing on an attached rope. Twenty-three adult male rhesus monkeys were used to evaluate these two methods. After a period of adaptation, saliva samples were collected from 21 of 23 subjects. Saliva collection was faster with the pole than with the screen method (P < 0.01), but the pole method was not suitable for some animals because of their tendency to bite off the attached rope. An analysis of 19 saliva samples revealed a mean cortisol concentration of 0.84 microg/dl (range 0.27-1.77 microg/dl). There was no statistically significant difference in cortisol value between methods used (P > 0.22). The influence of the flavoring on the cortisol assay was tested, and was found to have no significant effect (P > 0.28). Our results indicate that either technique can be used to safely collect saliva from unrestrained adult monkeys. Choice of technique will depend on the proclivities of individual monkeys.     

Body size and allometric variation in facial shape in children

Objectives

In group‐living primates, it has been reported that the alpha male exhibits high concentrations of cortisol and testosterone in the context of mating competition. We investigated how the presence of females affected salivary cortisol and testosterone levels in males from a small captive group of chimpanzees (Pan troglodytes). Specifically, we assessed whether the presence of females resulted in a rapid increase in salivary cortisol and testosterone levels in the alpha male.

Materials and methods

We compared the social behavior and salivary hormone concentrations of four males before and after the presentation of receptive females. Three times a day, we collected saliva samples, a useful matrix for investigating short‐term hormonal changes, and measured cortisol and testosterone concentration by liquid chromatography–tandem mass spectrometry (LC–MS/MS).

Results

The frequency of inter‐male aggression increased in the presence of females, indicating intense competition among males. Salivary cortisol levels increased in all males in the presence of females; however, the increase was significantly more pronounced in the alpha male. We found a complex three‐way interaction among the presence of females, sampling timings, and male dominance rank in the analysis of salivary testosterone. Contrary to our prediction, a post hoc analysis revealed that salivary testosterone levels decreased after female introduction and that the alpha male did not show a higher level of salivary testosterone.

Conclusions

Our study provides experimental evidence suggesting that the presence of females plays a significant role in the rank‐related variation in the cortisol levels in male chimpanzees. Furthermore, our findings demonstrate the usefulness of salivary hormones for detecting short‐term physiological changes in studies of socioendocrinology.

     

Salivary concentration of progesterone and cortisol significantly differs across individuals after correcting for blood hormone values

Between‐individual variation of salivary progesterone (P4) and cortisol levels does not always closely reflect blood hormone concentrations. This may be partly a function of individual differences in salivary hormone excretion. We tested whether time of day at sampling and ethnicity contributed to individual variation in salivary hormones after adjusting for blood hormone levels. Forty‐three Caucasian and 15 Japanese women (18–34 years) collected four sets of matched dried blood spot (DBS) and saliva specimens across a menstrual cycle (N = 232 specimen sets). Linear fixed‐effects (LFE) models were used to estimate the effects of diurnal variation and ethnicity on salivary P4 and cortisol while adjusting for DBS levels. For each hormone, women with exclusively positive or negative residuals (unexplained variance) from the LFE models were categorized as high‐ or low‐saliva‐to‐DBS hormone ratio (SDR; high or low salivary secretors), respectively. We found that salivary P4 (P < 0.05) was significantly higher in early morning compared to the afternoon, after controlling for DBS levels, ethnicity, and BMI. After further adjusting for this diurnal effect, significant individual variation in salivary P4 and cortisol remained: sixteen and nine women, respectively were categorized as low or high salivary secretors for both hormones (P < 0.001), suggesting systematic individual‐specific variation of salivary hormonal concentration. We conclude that when saliva is used to quantify P4 or cortisol levels, time of day at sampling should be controlled. Even with this adjustment, salivary P4 and cortisol do not closely mirror between‐ individual variation of serum P4 and cortisol in a substantial proportion of individuals. Am J Phys Anthropol 149:231–241, 2012. © 2012 Wiley Periodicals, Inc.     

Saliva and serum cortisol dynamics following intravenous dexamethasone in normal volunteers

In order to further explore the utility of saliva cortisol as an accurate measure of adrenal steroid production, dexamethasone (DEX) and saline were administered intravenously at 0800h to eight normal male volunteers in a randomized design, and the effects on serum and saliva cortisol concentrations were measured at hourly intervals from 0800h-2300h. Saliva cortisol values were highly correlated to serum cortisol levels within-subjects under both conditions (r = 0.78, p less than 0.025), however correlations were reduced in the DEX day sample pairs. Across-subject correlations at each time point were considerably more variable, reflecting interindividual differences in the saliva to serum cortisol ratios. No consistent time lag of saliva cortisol values in response to serum cortisol fluctuations was observed. These data suggest that saliva cortisol is an excellent index of changes in adrenal production of cortisol over time within individuals; however, it also suggests that salivary cortisol measures have less usefulness in comparing values across groups of individuals.     

Glucocorticoid response to exercise as measured by serum and salivary cortisol

Serum and salivary cortisol concentrations were studied in 78 elite athletes engaged in different sports, by subjecting them to high-intensity laboratory exercise. The mean difference in the pre-exercise cortisol concentrations in the seven groups studied were more marked in serum (from 311 to 768 nmol.l-1) than in saliva (from 17.9 to 22.7 nmol.l-1, only one group reaching 40 nmol.l-1). Judging from the correlation coefficients based on total variances, the post-/pre-exercise differences in cortisol concentrations in serum depended chiefly on pre-exercise values, while those in saliva tended to depend more on the postexercise concentrations. The coefficients of correlation between that difference and either the pre- or postexercise values were -0.71 and 0.47, respectively, for serum, and -0.51 and 0.58, respectively, for saliva. This would suggest that salivary cortisol concentration might be a more suitable variable for assessing glucocorticoid activity in exercise than serum cortisol concentration, probably being less sensitive to pre-exercise emotional state.     

Bacteria in the oral mucosa and its effects on the measurement of cortisol, dehydroepiandrosterone, and testosterone in saliva

Bacteria load in saliva was experimentally manipulated, and the consequences for the measurement of salivary testosterone (T), dehydroepiandrosterone (DHEA), and cortisol (C) were examined. Healthy adults (n = 19) donated the first saliva sample upon rising after which they rinsed their mouths with water, waited 10 min, and donated a second sample. Samples were either left untreated or passed through a 0.22-microm filter and then frozen at -80 degrees C or incubated at room temperature (RT) for 10 days. Aliquots of each sample were cultured on agar to determine baseline and post-incubation (or freezing) bacteria load. Bacteria counts were not significantly influenced by rinsing (with water), were substantially reduced by filtration, and increased by incubation at RT. Average levels of salivary T and C, but not DHEA, were significantly lower in samples stored at RT than samples frozen the day of collection. The change in bacteria count induced by storing samples at RT was associated with a change in testosterone but not cortisol or DHEA. When samples were passed through a 0.22-microm filter bacteria counts were reduced, and the association between bacteria and testosterone was reduced to non-significant. These findings contribute to a growing body of literature revealing that the process of sample collection, storage, and handling can dramatically influence the accuracy of information generated when salivary biomarkers are integrated into research and clinical diagnostics.     

Salivary and plasma cortisol response to adrenocorticotropin administration in pigs

Two experiments were conducted to determine the responsiveness of salivary and plasma cortisol to acute (i.v.), depot (i.m.) and chronic (repeated i.m.) adrenocorticotropin (ACTH) administration in swine. In Experiment 1, barrows (castrated pigs) were assigned to one of three injection treatments: (1) saline i.m. (SHAM1, n=2); (2) 0.75 IU/kg BW ACTH in saline i.v. (ACUTE, n=2); (3) 2.25 IU/kg BW ACTH in gel i.m. (DEPOT, n=3). Total cortisol concentrations were determined for concurrent saliva and blood samples. Correlations between salivary and plasma cortisol within treatments were: SHAM1, r=0.60; ACUTE, r=0.58; DEPOT, r=0.79. In Experiment 2, barrows were assigned to one of two injection treatments: (1) gel i.m. (SHAM2, n=3); (2) 2.25 IU/kg BW ACTH in gel i.m. (CHRONIC, n=4). The injections occurred every 6 h for a total of eight injections. Concurrent saliva and blood samples were obtained every 3 h for 72 h followed by an increasing sampling interval until day 6. Overall correlations between salivary and plasma cortisol were: SHAM2, r=0.30 and CHRONIC, r=0.61. Experiment 1 found that the relationship between salivary and plasma cortisol was stronger during longer (DEPOT) than shorter (ACUTE) ACTH stimulation. Experiment 2 found a strong relationship between the two measurements during chronic ACTH stimulation, but that relationship weakened after ACTH stimulation ceased.     

Gender as a determinant of responses to a self-screening questionnaire on anxiety and depression by patients with coronary artery disease

Background: Because patients with coronary artery disease (CAD) could benefit from interventions to decrease psychological distress, it is important to identify these individuals. Both salivary cortisol level and the Hospital Anxiety and Depression Scale (HADS) are recognized measures of stress/anxiety and depression.Objectives: This study was designed to determine whether there is an association between anxiety and depression, as measured by the HADS, and salivary cortisol levels among patients with CAD, and whether this association is affected by gender.Methods: All adult residents of Alberta, Canada, undergoing their first cardiac catheterization for CAD (≥50% occlusion of ≥2 coronary arteries) were eligible for participation in this study. A 14-question survey (the HADS) and 3 saliva-collection devices (a 1-day supply) were sent to the participants' home within 1 week of their initial cardiac catheterization. Participants were asked to take saliva samples for determination of cortisol levels on waking and at 30 and 60 minutes after waking, and then return the completed questionnaire and saliva samples using a prepaid express mailing envelope.Results: Seventy-one adults (52 men and 19 women) participated in the study. Mean (SD) ages were 68.4 (4.6) years for men and 69.1 (4.4) years for women. Among the women, significant negative correlations were found between the HADS anxiety score and the wake-up and 30-minute cortisol levels (higher HADS scores were associated with lower cortisol levels) (all, P < 0.05). Also among women, negative correlations were found between the HADS depression score and the salivary cortisol values, but the differences were not statistically significant. Conversely, among the men, nonsignificant positive correlations were found between the HADS anxiety scores and the salivary cortisol levels (higher HADS scores were associated with higher cortisol levels), and statistically significant positive correlations were observed between the HADS depression scores and all 3 salivary cortisol values (all, P < 0.05).Conclusions: Our findings suggest that the HADS is an appropriate screening instrument for anxiety and depression in patients with CAD. In particular, the scale appears to be sensitive for measuring anxiety in women and depression in men. When the HADS is used clinically as a screening tool, it should be examined through a “gender-based lens.”.     

Electronic monitoring of salivary cortisol sampling compliance in daily life

Naturalistic research methods have been developed to collect data in the daily environment, providing ecological valid measures. Recent reports suggest, however, that compliance with fixed time sampling protocols may be problematic and can bias results. This study investigated compliance with an intensive, random time sampling protocol for salivary cortisol and effects of non-compliance on cortisol results. Twenty female twin pairs and nineteen of their sisters were instructed to take saliva samples when signaled at ten unpredictable moments on each of five consecutive days. Subjects recorded collection times, unaware that compliance with the sampling protocol was being investigated by means of electronic monitoring devices. Samples taken < or = 15 min after the signal, according to self-report, were defined as adherent to the protocol. Samples taken < or = 10 min after the self-reported collection time, according to the monitor, were defined as accurate. Self-reported adherence to the sampling protocol was 96.4%. Verified compliance was somewhat lower, with 81% of all saliva samples accurately timed. Contrary to previous reports, inclusion of non-compliant samples in the analysis did not distort the cortisol diurnal profile. Intensive, random time sampling appears to have advantages over fixed time sampling for obtaining valid cortisol profiles when researchers do not have devices to monitor compliance. Results indirectly support the validity of momentary self-report data about daily experiences obtained with the same sampling methods.