Seasonal Variation in Human Salivary Cortisol Concentration

Measurement of cortisol concentration can contribute important information about an individual's ability to adjust to various environmental demands of both physical and psychosocial origin. However, one uncertainty that affects the possibilities of correctly interpreting and designing field studies is the lack of observations of the impact of seasonal changes on cortisol excretion. For this reason, the month‐to‐month changes in diurnal cortisol concentration, the awakening cortisol response (ACR), maximum morning concentration, and fall during the day were studied in a group of 24 healthy men and women 32 to 61 yrs of age engaged in active work. On one workday for 12 consecutive months, participants collected saliva at four time points for determination of cortisol: at awakening, +30 min, +8 h, and at 21:00 h. Data were analyzed by a repeated measures design with month (12 levels) and time‐of‐day (4 levels) as categorical predictors. Cortisol concentrations were analyzed on a log scale. The diurnal pattern of cortisol was similar across months (interaction between month and time of day: p>0.4). The main effects of month and time‐of‐day were statistically significant (p <0.001). Highest concentrations were observed in February, March, and April, and lowest concentrations were observed in July and August. There were no statistically significant effects in any of the other measures, or between men and women. In conclusion, a seasonal variation in salivary cortisol concentrations was detected in an occupationally active population. Thus, seasonal variation needs to be taken into account when designing and evaluating field studies and interventions and when making comparisons across studies.     

Diurnal variation of salivary cortisol in captive African elephants (Loxodonta africana) under routine management conditions and in relation to a translocation event

The present study assessed the diurnal variation in salivary cortisol in captive African elephants during routine management (baseline) and in relation to a potential stressor (translocation) to evaluate to what extent acute stress may affect diurnal cortisol patterns. Under baseline conditions, we collected morning and afternoon saliva samples of 10 animals (three zoos) on different days in two study periods (n = 3–10 per animal, daytime and period). Under stress conditions, we sampled the transported cow (newcomer) and the two cows of the destination zoo before and after the transport in the morning and afternoon (n = 3–9 per animal, daytime and transport phase), as well as after the first introduction of the newcomer to the bull (n = 1 per animal). Cortisol was measured in unextracted samples by enzyme immunoassay. Under baseline conditions, we observed the expected diurnal variation with higher cortisol levels in the morning than in the afternoon. Under stress conditions, neither a significant difference between pre- and posttransport, nor between morning and afternoon levels was found. The percentage difference between morning and afternoon cortisol after the transport, however, was remarkably lower than before the transport in the newcomer potentially indicating a stress response to familiarization. Saliva samples taken immediately after the introduction of the newcomer to the bull revealed a marked cortisol increase. Our findings indicate that stressors may disturb the diurnal cortisol rhythm. Furthermore, provided that samples can be collected promptly, salivary cortisol is a useful minimally invasive measure of physiological stress in the African elephant.     

Diurnal salivary cortisol is associated with body mass index and waist circumference: The multiethnic study of atherosclerosis

Objective:

Neuroendocrine abnormalities, such as activation of the hypothalamic‐pituitary‐adrenal (HPA) axis, are associated with obesity; however, few large‐scale population‐based studies have examined HPA axis and markers of obesity. We examined the cross‐sectional association of the cortisol awakening response (CAR) and diurnal salivary cortisol curve with obesity.

Design and Methods:

The Multiethnic Study of Atherosclerosis Stress Study includes 1,002 White, Hispanic, and Black men and women (mean age 65 ± 9.8 years) who collected up to 18 salivary cortisol samples over 3 days. Cortisol profiles were modeled using regression spline models that incorporated random parameters for subject‐specific effects. Cortisol curve measures included awakening cortisol, CAR (awakening to 30‐min postawakening), early decline (30 min to 2‐h postawakening), late decline (2‐h postawakening to bedtime), and the corresponding areas under the curve (AUC). Body mass index (BMI) and waist circumference (WC) were used to estimate adiposity.

Results:

For the entire cohort, both BMI and WC were negatively correlated with awakening cortisol (P < 0.05), AUC during awakening rise, and early decline and positively correlated to the early decline slope (P < 0.05) after adjustments for age, race/ethnicity, gender, diabetes status, socioeconomic status, β‐blockers, steroids, hormone replacement therapy, and smoking status. No heterogeneities of effects were observed by gender, age, and race/ethnicity.

Conclusions:

Higher BMI and WC are associated with neuroendocrine dysregulation, which is present in a large population sample, and only partially explained by other covariates.     

Quantifying blood leakage into the oral mucosa and its effects on the measurement of cortisol, dehydroepiandrosterone, and testosterone in saliva

The impact of blood leakage due to microinjury to the oral cavity on the measurement of salivary hormones was examined. Saliva samples were collected before, immediately after, and then every 15 min for 1 h following vigorous tooth brushing. Blood in saliva was quantified by visual inspection of discoloration, Hemastix reagent strips to detect hemoglobin, and an immunoassay for transferrin. The presence of blood in saliva immediately after microinjury was confirmed by all methods. Hemoglobin and transferrin levels remained elevated over baseline for at least 30 min. Levels of salivary testosterone increased over baseline and remained elevated for 30 min in response to microinjury. Microinjury induced change in salivary testosterone was more closely associated with the change in transferrin than hemoglobin levels or discoloration ratings. On average, levels of salivary dehydroepiandrosterone (DHEA) did not increase in response to microinjury. However, individual differences in microinjury induced change in DHEA were associated with discoloration ratings. Salivary cortisol levels, on average, were neither responsive to microinjury, nor were individual differences in cortisol change associated with blood contamination measures. Neither diurnal nor gender-related differences in baseline hormone levels predicted the impact of blood leakage on quantitative salivary measurements. The findings suggest ecologically valid minor-to-moderate level microinjuries to the oral cavity have negligible effects on the measurement of salivary cortisol, but may be important to quantify and control when assessing other hormones especially testosterone.     

Circadian Rhythm of Salivary Cortisol in Asian Elephants (Elephas maximus): A Factor to Consider During Welfare Assessment

Elevated glucocorticoid levels during an extended time period might be a stress indicator in nonhuman animals. Therefore, knowledge of the circadian pattern of cortisol secretion is very important to correctly interpret data obtained for welfare assessment of animals in captivity through salivary cortisol. In order to define the circadian rhythm of salivary cortisol secretion in the Asian elephant (Elephas maximus), morning and evening saliva samples of 3 Asian elephants were collected and analyzed by radioimmunoassay. Significantly higher salivary cortisol concentrations were found in the morning than in the evening in all individuals. These results show that salivary cortisol of Asian elephants follows a diurnal pattern of secretion, which could be taken into account when using this methodology to assess welfare in captive Asian elephants.     

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.

     

Increased Cortisol Bioavailability,Abdominal Obesity,and the Metabolic Syndrome in Obese Women

Objective: This study was conducted to obtain a detailed profile of hypothalamo‐pituitary‐adrenal (HPA) axis activity and reactivity and its differential relationships with body fat distribution and total fat mass in premenopausal obese women. Research Methods and Procedures: Cortisol responses to stimulation (awakening, food intake, exercise) and suppression (0.25 mg dexamethasone), cortisol metabolism, and tissue sensitivity to glucocorticoids were studied in 53 premenopausal obese women grouped according to their waist‐to hip ratio: women with abdominal body fat distribution (A‐BFD; n = 31) and women with peripheral fat distribution (P‐BFD; n = 22). Results: Comparatively, A‐BFD women had 1) lower awakening salivary cortisol levels; 2) increased salivary responsiveness to a standardized lunch; 3) similar pituitary sensitivity to dexamethasone but decreased sensitivity of monocytes to dexamethasone; 4) similar 24‐hour urinary free cortisol but increased 24‐hour urinary ratio of cortisone‐to‐cortisol; and 5) no difference in corticosteroid binding protein parameters. Discussion: Although abdominal obesity is not very different from generalized obesity in terms of HPA function, subtle variations in HPA axis activity and reactivity are evidenced in A‐BFD premenopausal obese women.     

Individual,social, and environmental factors affecting salivary and fecal cortisol levels in captive pied tamarins (Saguinus bicolor)

Pied tamarins (Saguinus bicolor) are endangered New World primates, and in captivity appear to be very susceptible to stress. We measured cortisol in 214 saliva samples from 36 tamarins and in 227 fecal samples from 27 tamarins, and investigated the effects of age, sex, pregnancy, rearing history, social status, weight, group composition, and enclosure type using generalized linear mixed models. There was no effect of age on either fecal or salivary cortisol levels. Female pied tamarins in late pregnancy had higher fecal cortisol levels than those in early pregnancy, or nonpregnant females, but there was no effect of pregnancy on salivary cortisol. Females had higher salivary cortisol levels than males, but there was no effect of rearing history. However, for fecal cortisol, there was an interaction between sex and rearing history. Hand‐reared tamarins overall had higher fecal cortisol levels, but while male parent‐reared tamarins had higher levels than females who were parent‐reared, the reverse was true for hand‐reared individuals. There was a trend towards lower fecal cortisol levels in subordinate individuals, but no effect of status on salivary cortisol. Fecal but not salivary cortisol levels declined with increasing weight. We found little effect of group composition on cortisol levels in either saliva or feces, suggesting that as long as tamarins are housed socially, the nature of the group is of less importance. However, animals in off‐show enclosures had higher salivary and fecal cortisol levels than individuals housed on‐show. We suggest that large on‐show enclosures with permanent access to off‐exhibit areas may compensate for the effects of visitor disturbance, and a larger number of tamarins of the same species housed close together may explain the higher cortisol levels found in tamarins living in off‐show accommodation, but further research is needed.     

Specific gravity and creatinine as corrections for variation in urine concentration in humans,gorillas, and woolly monkeys

Hormones excreted in the urine are widely used to assess the physiological and psychological condition of unrestrained animals. In order to control for variation in the water concentration of urine samples, the hormone concentration is often indexed to the concentration of creatinine. Because there are several problems with using creatinine, we have investigated the efficacy of specific gravity as an alternative basis for adjusting the hormone concentration in humans, gorillas, and woolly monkeys. In an experimental manipulation of human urine hydration, ten volunteers drank a water load proportional to body weight, and provided complete urine collection and saliva samples for four consecutive 20 min intervals. From the urine, we measured cortisol (radioimmunoassay), creatinine (colorimetric assay), and specific gravity (refractometer). Only cortisol was assayed from saliva. During 80 min following water ingestion, cortisol, creatinine, and specific gravity declined as urine became diluted; however, total cortisol excretion remained constant. Only cortisol concentration indexed to specific gravity accurately reflected the consistent cortisol excretion. Specific gravity and creatinine‐corrected cortisol values were highly correlated but were significantly different. Salivary cortisol provided evidence for the relative stability of serum cortisol. To determine the utility of these corrections in other primates, we compared specific gravity‐ and creatinine‐corrected cortisol in urine samples from captive gorillas (N=16) and woolly monkeys (N=8). As with the human study, the two corrections were strongly correlated in each species, but the means were different. Specific gravity correction was superior in revealing the circadian variation in cortisol. Am. J. Primatol. 72:1082–1091, 2010. © 2010 Wiley‐Liss, Inc.     

Validation of salivary cortisol and testosterone assays in chimpanzees by liquid chromatography‐tandem mass spectrometry

Owing to its high temporal sensitivity, saliva has distinct advantages for measuring steroids, compared with other noninvasive samples such as urine and feces. Here, we report the validity of assaying salivary cortisol (C) and testosterone (T) using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) in captive male chimpanzees, Pan troglodytes. For both the C and T concentrations, we found positive relationships between saliva and plasma. The concentrations of C and T in saliva showed clear patterns of diurnal fluctuation, whereas those in urine and feces did not. These results suggest that the salivary steroid concentrations can be regarded as good indicators of circulating steroid levels. We also developed and validated an efficient method for collecting saliva samples from cotton rope. Although rope includes inherent steroid‐like compounds and may affect the accuracy of steroid measurements, our rope‐washing procedures effectively removed intrinsic steroidal materials. There was a significant association between the C and T concentrations measured from saliva collected from rope licked by the chimpanzees and those measured from saliva collected directly from the mouth. Salivary T values estimated by LC/MS‐MS were similar to those measured by radioimmunoassay. The results indicate the usefulness of saliva as a noninvasive steroid measure and that steroids in the saliva of chimpanzees can be accurately measured by LC‐MS/MS. Am. J. Primatol. 71:696–706, 2009. © 2009 Wiley‐Liss, Inc.     

Circadian rhythms of salivary cortisol content during long-term space flight

Adaptation mechanisms of adrenal function related to secretion of cortisol were studied under conditions of microgravity. Parameters of diurnal rhythms of salivary cortisol were studied by Russian cosmonauts on board orbital station Mir during long-term space flights (SF). The preflight circadian rhythms of salivary cortisol in cosmonauts were characterized by the morning maximum occurring at 9∶43 a.m., the fluctuation amplitude 6.05 nmol/1, and the daily average concentration 8.79 nmol/l. The characteristics of cortisol diurnal rhythm changed under conditions of long-term space flight. On average, the rhythm measure and amplitude decreased after two months of flight. The postflight maximum concentration of free cortisol tended to occur later in the day. Evidently, the motor activity during SF, i.e., prophylactic exercises along with other factors, significantly influenced the parameters of cortisol circadian rhythm that was revealed by the individual variability of findings during the flight. After the long-term SF, individual ratios of salivary and plasma cortisol levels increased against the background of increased plasma content of the hormone, i.e., the fraction of free, physiologically active hormone in the total pool of circulating molecules decreased.     

Relationship of stimulated whole saliva cortisol level with the severity of a feeling of dry mouth in menopausal women

doi: 10.1111/j.1741‐2358.2010.00403.x Relationship of stimulated whole saliva cortisol level with the severity of a feeling of dry mouth in menopausal women Objectives: To evaluate the relationship of stimulated whole saliva cortisol level with the severity of a feeling of dry mouth (DM) in menopausal women. Background: A feel of DM is a major complaint for many elderly individuals and strongly associated with the menopause. The exact mechanisms that mediate sensation of DM in menopausal women have not been firmly established. Methods: A case–control study was carried out on 104 selected menopausal women with/without a feeling of DM, conducted at the Clinic of Oral Medicine, Tehran University of Medical Sciences. Xerostomia Inventory (XI) score was used as an index of DM severity. Stimulated whole saliva cortisol concentration (stimulated by chewing standard‐sized paraffin for 60 s) was measured by ELISA. Statistical analysis by Student’s t‐test and Spearman correlation was used. Results: The mean cortisol concentration of saliva, but not saliva cortisol output, was significantly higher in the cases than in the controls. There was significant positive correlation between XI score and concentration (r = 0.357, p = 0.000) or output (r = 0.223, p = 0.017) of stimulated whole saliva cortisol. Conclusions: It appears that stimulated whole saliva cortisol is high in menopausal women with a feeling of DM.     

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.     

Inhibitory effect of saliva from secretors and non-secretors on binding of meningococci to epithelial cells

Abstract Carriage of Neisseria meningitidis B:4:P1.15 was higher among non-secretors during a school outbreak of meningitis; non-secretors had lower levels of anti-meningococcal salivary IgM. Flow cytometry was used to assess effects of secretor and non-secretor saliva on binding of B:4:P1.15 to buccal epithelial cells: (1) to assess inhibition by IgA and IgM; and (2) to assess contributions of salivary antibodies to inhibitory activities. Greater inhibition was obtained with secretor saliva: pooled ( P = 0.049); fresh ( P = 0.0001). Purified IgA ( P = 0.02) and IgM ( P = 0.03) were equally inhibitory. After absorption of anti-meningococcal antibodies, there was still significant inhibitory activity in the pools: secretors ( P = 0.018); non-secretors ( P = 0.005). These results indicate that both secretory immunoglobulins and other factors contribute to protection against colonisation by meningococci and might explain the increased carriage of B:4:P1.15 in this population.     

Differences in saliva collection location and disparities in baseline and diurnal rhythms of alpha-amylase: a preliminary note of caution

Identified in the early 1980s as a surrogate marker of the sympathetic nervous system component of the stress response, there has been renewed interest in measuring salivary alpha-amylase (sAA) to test biosocial models of stress vulnerability. This brief report presents studies that document that oral fluids from the parotid and submandibular gland areas had higher sAA values than did whole saliva specimens, and sAA values in whole saliva were higher than levels measured in oral fluids from the sublingual gland area. sAA in oral fluids from the parotid and submandibular gland areas showed the highest and more pronounced diurnal variation than levels in whole saliva, and sAA in sublingual saliva showed the lowest and shallowest diurnal variation. When this source of inherent variability in sAA activity levels is not controlled for by collecting oral fluids consistently from specific gland areas, the detection of individual differences, associations between sAA and “behavioral” variables, and intra-individual change in sAA levels may be compromised. Awareness, and management, of this ubiquitous source of measurement error in sAA are essential to ensure the success of future research on the correlates and concomitants of sAA levels, stress-related reactivity and recovery, and diurnal variation.     

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.     

Noninvasive technique for the repeated sampling of salivary free cortisol in awake,unrestrained squirrel monkeys

The use of noninvasive measures of hypothalamic-pituitary-adrenal (HPA) axis function is of growing interest among preclinical and clinical investigators. This report describes a method for the repeated assessment of salivary free cortisol in awake, unrestrained squirrel monkeys (Saimiri sciureus) based on a saliva sampling technique previously developed for rhesus monkeys. Individually housed adult male squirrel monkeys were trained to chew on dental rope attached to a pole, from which saliva was extracted by centrifugation and analyzed for cortisol by radioimmunoassay (RIA). Eight of nine monkeys readily acquired the task, reliably providing adequate saliva samples for the assay. Salivary free cortisol levels were examined in these subjects under basal conditions and in response to two types of neuroendocrine challenge. Levels of salivary free cortisol showed relatively low intra- and interindividual variability, with mean individual morning levels ranging between 17.1 and 37.9 microg/dl. Squirrel monkeys demonstrated a consistent daily rhythm in salivary free cortisol ranging from a high of 27.4 +/- 5.2 microg/dl (mean +/- SEM) at 12 P.M. to a low of 7.5 +/- 1.6 microg/dl at 6 P.M. Intravenous (IV) challenges with 1 microg/kg ACTH, or 10 and 50 microg/kg CRF resulted in significant increases in salivary free cortisol. The described sampling technique provides a reliable and sensitive means for repeated measurement of HPA activity in unrestrained, awake squirrel monkeys. In addition, our findings illustrate several features of HPA system rhythmicity and reactivity using salivary cortisol instead of blood plasma or serum.     

Diurnal rhythms of plasma growth hormone,somatostatin, thyroid hormones,cortisol and glucose concentrations in rainbow trout,Oncorhynchus mykiss,during progressive food deprivation

Abstract

The diurnal patterns of changes in plasma cortisol, growth hormone (GH), somatostatin‐14 (SRIF), thyroid hormones (L‐thyroxine, T₄ and triiodo‐L‐thyronine, T₃) and glucose were investigated in rainbow trout, Oncorhynchus mykiss, both during single meal‐feeding and during a progressive fast of 13 weeks. All measured variables exhibited a diurnal pattern in fed rainbow trout, most of which appeared to be correlated with the time of feeding (30–60 min after the onset of light), while additional changes, associated with the scotophase were also found for cortisol. Although fasting had no affect on mean daily plasma cortisol or SRIF concentrations, there was a progressive increase in mean daily plasma GH concentrations and a progressive decrease in mean daily plasma thyroid hormone and glucose concentrations associated with fasting. However, for GH, significant changes were not evident until week 10 of the fast. In addition, fasting appeared to phase‐shift the diurnal patterns of plasma GH, cortisol and glucose concentrations; the consequence of such a shift is that conclusions as to the effects of fasting, if based on a single time of sampling, may be flawed.     

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.”.