Comparative analysis of salivary cortisol measurements using different assay methods in relation to serum-free cortisol measurement.

Objectives: Salivary cortisol reflects the biologically active form of serum cortisol, offering a noninvasive evaluation method for the diurnal rhythm of the hypothalamic-pituitary-adrenal (HPA) axis. While liquid chromatography-tandem mass spectrometry (LC-MS/MS) is known for its specificity, immunoassays (IA) are commonly used because of their simplicity. This study aimed to assess the performance of salivary cortisol measurement using both IA and LC-MS/MS in comparison to serum-free cortisol measurement. Methods: Assay results for 188 saliva and 94 serum samples from 47 participants were analyzed. Salivary samples collected at different time points were analyzed using IA and LC-MS/MS. Serum samples were analyzed for cortisol, cortisol-binding globulin, and free cortisol. The statistical analyses included correlations and method comparisons. Results: The diurnal salivary cortisol profiles exhibited a comparable circadian rhythm pattern; however, the concentrations measured using IA were consistently higher than those measured using LC-MS/MS. The correlation analysis revealed robust associations among salivary cortisol (IA), salivary cortisol (LC-MS/MS), and serum-free cortisol levels (LC-MS/MS). However, the method comparison revealed a systematic bias between IA and LC-MS/MS in salivary cortisol measurement. Conclusions: This study contributes to the ongoing debate on assay techniques by affirming the suitability of IA and LC-MS/MS for salivary cortisol measurement to assess dynamic changes in HPA axis activity. The identified systematic bias emphasizes the importance of selecting methods based on specific research or clinical requirements.

Exploratory Clinical Trial of a Depression Diagnostic Software That Integrates Stress Biomarkers and Composite Psychometrics.

OBJECTIVE: This study evaluated the clinical effectiveness of Minds.NAVI, a depression screening kit combining psychometric measures and stress hormone biomarkers, in a prospective clinical trial. The objective was to assess its potential as a depression screening tool and investigate the associations between psychological assessments, salivary hormone staging, and depression severity. METHODS: Thirty-five participants with major depressive disorder and 12 healthy controls (HCs) were included. The Minds.NAVI software, utilizing the PROtective and Vulnerable factors battEry Test (PROVE) and salivary cortisol/dehydroepiandrosterone (DHEA) analysis, was employed. The PROVE test is a comprehensive self-report questionnaire that assesses depressive symptoms, suicide risk, attachment style, adverse childhood experiences, mentalization capacity, and resilience. In addition, salivary cortisol and DHEA levels were measured to evaluate the functional stage of the hypothalamic-pituitary-adrenal (HPA) axis. RESULTS: Minds.NAVI exhibited 100% sensitivity, 91.7% specificity, and 97.9% accuracy in distinguishing depression from HCs within an exploratory small group. Salivary stress hormone phases showed changes with depression stage (p=0.030), and the proportion of patients with "adrenal exhaustion stage" was higher in the moderate/severe depression group (p=0.038). Protective/vulnerable factors differed significantly between controls and depressed groups (p<0.001). Cortisol awakening response inversely correlated with depressive symptom severity (r=-0.31, p=0.034). CONCLUSION: This study suggested possible clinical effectiveness of Minds.NAVI, a depression screening tool that integrates psychometric measures and stress hormone biomarkers. The findings support the potential association between depression, chronic stress, and HPA axis hyporesponsiveness.

Relationship of Resilience Factors With Biopsychosocial Markers Using a Comprehensive Home Evaluation Kit for Depression and Suicide Risk: A Real-World Data Analysis.

Objectives: Adverse childhood experiences (ACEs) are fundamental factors in developing depression with increased suicide risk. Resilience is considered an important protective factor that can prevent trauma survivors from developing depression. We developed a home evaluation kit for a comprehensive assessment of bio-psycho-social factors related to depression and suicide. This kit contained a psycho-social evaluation battery, named the Protective and Vulnerable factors battery questionnaire (PROVE) comprising depressive symptoms and suicide risk, as well as various depression-related psychosocial factors, such as ACE, resilience, mentalization capacity, and attachment, via online survey tools. Furthermore, salivary cortisol levels were used as biological indicators to assess the hypothalamus-pituitary-adrenal axis function. Methods: Real-world data analysis was made out of data collected from participants who visited CHEEU Counseling center or Gangnam Severance hospital for mental health check-ups. The participants were put into three mental state groups (green-normal, yellow-borderline, and red-risk) depending on the result of PROVE battery. The difference between psychosocial factors and salivary cortisol indicators by the group was identified by analysis of covariance with sex and age as covariates. Linear regression analysis was conducted to find a significant association of resilience score with other bio-psycho-social variables, such as ACE, attachment, mentalization, or post-awakening cortisol concentrations (area under the curve with respect to ground, AUCg). A partial correlation analysis was performed to evaluate the relationship of AUCg with psychosocial factors. Results: Depression-related psycho-social indicators were significantly different among groups. Insecure attachment and the mentalization problem are negatively influencing factors to resilience. Furthermore, the severity of depression in participants with ACE was also influenced by mentalization problems. AUCg was different according to the PROVE group, presence of ACE, or resilience level. In addition, AUCg showed a positive correlation with resilience score but negative correlations with depressive symptoms, ACE, mentalization problems, and anxiety or avoidance attachment. Conclusion: This study suggests that there are some key factors negatively affecting resilience: insecure attachment and mentalization problems. In groups with ACE, a mentalization problem was suggested as a factor that can increase depressive symptoms. AUCg was associated with resilience as well as several other vulnerable factors of depression, showing its potential as a promising biomarker.

Virtual reality-based monitoring test for MCI: A multicenter feasibility study.

Objectives: As the significance of the early diagnosis of mild cognitive impairment (MCI) has emerged, it is necessary to develop corresponding screening tools with high ecological validity and feasible biomarkers. Virtual reality (VR)-based cognitive assessment program, which is close to the daily life of the older adults, can be suitable screening tools for MCI with ecological validity and accessibility. Meanwhile, dehydroepiandrosterone (DHEA) has been observed at a low concentration in the older adults with dementia or cognitive decline, indicating its potential as a biomarker of MCI. This study aimed to determine the efficacy and usability of a VR cognitive assessment program and salivary DHEA for screening MCI. Methods: The VR cognitive assessment program and the traditional Montreal Cognitive Assessment (MOCA) test were performed on 12 patients with MCI and 108 healthy older adults. The VR program operates in a situation of caring for a grandchild, and evaluates the memory, attention, visuospatial, and executive functions. An analysis of covariance (ANCOVA), a partial correlation analysis, and receiving operating characteristic (ROC) curve analysis were conducted for statistical analysis. Results: According to the ANCOVA, no significant difference in MOCA scores was found between the normal and MCI groups (F = 2.36, p = 0.127). However, the VR total score of the MCI group was significantly lower than that of the normal group (F = 8.674, p = 0.004). There was a significant correlation between the MOCA and VR scores in the total and matched subdomain scores. The ROC curve analysis also showed a larger area under the curve (AUC) for the VR test (0.765) than for the MOCA test (0.598), and the sensitivity and specificity of the VR program were 0.833 and 0.722, respectively. Salivary DHEA was correlated with VR total (R 2 = 0.082, p = 0.01) and attention scores (R 2 = 0.086, p = 0.009). Conclusion: The VR cognitive test was as effective as the traditional MOCA test in the MCI classification and safe enough for older adults to perform, indicating its potential as a diagnostic tool. It has also been shown that salivary DHEA can be used as a biomarker for MCI.

Association of the HPA axis response to upcoming competition and shooting outcomes in elite junior shooting players.

Successful shooting performance in competition is reliant on several factors such as shooting techniques and competition-associated psychological stresses. This study examined the hypothalamus-pituitary-adrenal (HPA) axis response to upcoming competition and its association with shooting outcomes in elite junior shooting players. The cortisol awakening response (CAR) and dehydroepiandrosterone (DHEA) secretion after awakening were measured for two consecutive days (the day before and on the day of competition for the selection of national shooting team members) in 19 junior men and 21 junior women players, and the shooting scores of the individual players were obtained. The total cortisol secretion during the CAR period (CARauc) increased, but total DHEA secretion during the post-awakening period (Daucawk) decreased on the day of competition, compared with one day before competition. The CARauc was higher in women than in men players, whereas Daucawk was higher in men than in women players across the two consecutive days. Cortisol and DHEA levels were comparable between low-scored (below the mean scores for air pistol or air rifle players) and high-scored players one day before competition. However, the CARauc on the day of competition was higher and the variations in the CARauc and molar CARauc/Daucawk ratios across the two consecutive days were greater in low-scored than in high-scored men and women players. These results indicated that upcoming competition involves alterations of the CAR and DHEA secretion after the awakening period, and greater HPA response to the upcoming competition was adversely associated with shooting scores in junior shooting athletes.Lay summaryAn important upcoming competition was perceived as a strong stressor on awakening that induced alteration in CAR and DHEA secretion after the awakening period in elite shooting players. This study observed that, irrespective of their age and period of shooting practice, the HPA axis function on the day of competition was associated with shooting outcomes in elite shooting players.

Altered Hypothalamus-Pituitary-Adrenal Axis Function: A Potential Underlying Biological Pathway for Multiple Concurrent Symptoms in Patients With Advanced Lung Cancer.

OBJECTIVE: Patients with advanced cancer commonly experience multiple symptoms that present as groups or clusters. The present study aimed to examine whether hypothalamus-pituitary-adrenal (HPA) axis dysfunction underlies the concurrent multiple symptoms in patients with advanced cancer. METHODS: Patients' cortisol levels were determined in saliva samples collected after awakening (0, 30, and 60 minutes after awakening) and at nighttime (21:00-22:00 PM) from 46 patients with lung cancer (15.2% women), with a mean (standard deviation) age of 64.3 (9.2) years and 47 healthy participants (53.2% women; age = 62.0 [4.6] years). Cancer-related symptoms were measured using the M.D. Anderson Symptom Inventory (MDASI). RESULTS: Compared with healthy participants, patients showed a significantly reduced cortisol awakening response (F(1,364) = 46.2, p < .001) and had flatter diurnal slope of cortisol (larger ß values) (mean [standard error of the mean] = -0.64 [0.06] versus -0.18 [0.05], p < .001). Altered HPA axis function was significantly and adversely associated with performance status and burden of symptoms (all p values < .01). However, each MDASI item varied widely in the degree of association with the HPA axis function. Hierarchical clustering analysis based on Spearman's rank correlation with complete linkage identified that nausea was clustered with vomiting, numbness, and dry mouth, whereas the other nine MDASI core symptoms associated with altered HPA axis function were clustered together. CONCLUSIONS: Altered HPA axis function may be a possible biological pathway that can explain the concurrence of core symptoms in patients with advanced lung cancer.

Transforming growth factor-ß1 signaling represses testicular steroidogenesis through cross-talk with orphan nuclear receptor Nur77.

Transforming growth factor- ß1 (TGF-ß1) has been reported to inhibit luteinizing hormone (LH) mediated-steroidogenesis in testicular Leydig cells. However, the mechanism by which TGF-ß1 controls the steroidogenesis in Leydig cells is not well understood. Here, we investigated the possibility that TGF-ß1 represses steroidogenesis through cross-talk with the orphan nuclear receptor Nur77. Nur77, which is induced by LH/cAMP signaling, is one of major transcription factors that regulate the expression of steroidogenic genes in Leydig cells. TGF-ß1 signaling inhibited cAMP-induced testosterone production and the expression of steroidogenic genes such as P450c17, StAR and 3ß-HSD in mouse Leydig cells. Further, TGF-ß1/ALK5 signaling repressed cAMP-induced and Nur77-activated promoter activity of steroidogenic genes. In addition, TGF-ß1/ALK5-activated Smad3 repressed Nur77 transactivation of steroidogenic gene promoters by interfering with Nur77 binding to DNA. In primary Leydig cells isolated from Tgfbr2flox/flox Cyp17iCre mice, TGF-ß1-mediated repression of cAMP-induced steroidogenic gene expression was significantly less than that in primary Leydig cells from Tgfbr2flox/flox mice. Taken together, these results suggest that TGF-ß1/ALK5/Smad3 signaling represses the expression of steroidogenic genes via the suppression of Nur77 transactivation in testicular Leydig cells. These findings may provide a molecular mechanism involved in the TGF-ß1-mediated repression of testicular steroidogenesis.

Insulin directly regulates steroidogenesis via induction of the orphan nuclear receptor DAX-1 in testicular Leydig cells.

Testosterone level is low in insulin-resistant type 2 diabetes. Whether this is due to negative effects of high level of insulin on the testes caused by insulin resistance has not been studied in detail. In this study, we found that insulin directly binds to insulin receptors in Leydig cell membranes and activates phospho-insulin receptor-ß (phospho-IR-ß), phospho-IRS1, and phospho-AKT, leading to up-regulation of DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1) gene expression in the MA-10 mouse Leydig cell line. Insulin also inhibits cAMP-induced and liver receptor homolog-1 (LRH-1)-induced steroidogenic enzyme gene expression and steroidogenesis. In contrast, knockdown of DAX-1 reversed insulin-mediated inhibition of steroidogenesis. Whether insulin directly represses steroidogenesis through regulation of steroidogenic enzyme gene expression was assessed in insulin-injected mouse models and high fat diet-induced obesity. In insulin-injected mouse models, insulin receptor signal pathway was activated and subsequently inhibited steroidogenesis via induction of DAX-1 without significant change of luteinizing hormone or FSH levels. Likewise, the levels of steroidogenic enzyme gene expression and steroidogenesis were low, but interestingly, the level of DAX-1 was high in the testes of high fat diet-fed mice. These results represent a novel regulatory mechanism of steroidogenesis in Leydig cells. Insulin-mediated induction of DAX-1 in Leydig cells of testis may be a key regulatory step of serum sex hormone level in insulin-resistant states.

Phosphoenolpyruvate carboxykinase and glucose-6-phosphatase are required for steroidogenesis in testicular Leydig cells.

Cyclic AMP (cAMP) induces steroidogenic enzyme gene expression and stimulates testosterone production in Leydig cells. Phosphoenolpyruvate carboxykinase (PEPCK) is expressed in Leydig cells, but its role has not been defined. In this study, we found that PEPCK and glucose-6-phosphatase (Glc-6-Pase) are increased significantly following cAMP treatment of mouse Leydig cells. Moreover, cAMP treatment increased recruitment of the cAMP-response element-binding transcription factor and decreased recruitment of the corepressor DAX-1 on the pepck promoter. Furthermore, cAMP induced an increase in ATP that correlated with a decrease in phospho-AMP-activated protein kinase (AMPK). In contrast, knockdown or inhibition of PEPCK decreased ATP and increased phospho-AMPK. Treatment with an AMPK activator or overexpression of the constitutively active form of AMPK inhibited cAMP-induced steroidogenic enzyme promoter activities and gene expression. Liver receptor homolog-1 (LRH-1) was involved in cAMP-induced steroidogenic enzyme gene expression but was inhibited by AMPK activation in Leydig cells. Additionally, inhibition or knockdown of PEPCK and Glc-6-Pase decreased cAMP-mediated induction of steroidogenic enzyme gene expression and steroidogenesis. Finally, pubertal mouse (8-week-old) testes and human chorionic gonadotropin-induced prepubertal mouse testes showed increased PEPCK and Glc-6-Pase gene expression. Taken together, these results suggest that induction of PEPCK and Glc-6-Pase by cAMP plays an important role in Leydig cell steroidogenesis.

ERα/E2 signaling suppresses the expression of steroidogenic enzyme genes via cross-talk with orphan nuclear receptor Nur77 in the testes.

Estrogen receptor alpha (ERα) has been reported to affect steroidogenesis in testicular Leydig cells, but its molecular mechanism remains unclear. Here, we investigate the effect of estrogen and ERα on Nur77, a major transcription factor that regulates the expression of steroidogenic enzyme genes. In MA-10 Leydig cells, estradiol (E2) treatment, and interestingly ERα overexpression, suppressed the cAMP-induced and Nur77-activated promoter activity of steroidogenic enzyme genes via the suppression of Nur77 transactivation. ERα physically interacted with Nur77 and inhibited its DNA binding activity. In addition, ERα/E2 signaling decreased Nur77 protein levels. Consistent with the above results, the testicular testosterone level was higher in Leydig cell-specific ERα knock-out mice (ERα(flox/flox)Cyp17iCre) than in wild-type mice (ERα(flox/flox)). Taken together, these results suggest that ERα/E2 signaling controls the Nur77-mediated expression of steroidogenic enzyme genes in Leydig cells. These findings may provide a mechanistic explanation for the local regulation of testicular steroidogenesis by estrogenic compounds and ERα.

Cortisol awakening response and nighttime salivary cortisol levels in healthy working Korean subjects.

PURPOSE: Cortisol awakening response (CAR) and nighttime cortisol levels have been used as indices of adrenocortical activity. However, population-based statistical information regarding these indices has not been provided in healthy subjects. This study was carried out to provide basic statistical information regarding these indices. MATERIALS AND METHODS: Cortisol levels were measured in saliva samples collected immediately upon awakening (0 min), 30 min after awakening and in the nighttime on two consecutive days in 133 healthy subjects. RESULTS: We determined the mean [standard deviation (SD)], median (interquartile range) and 5th-95th percentile range for each measure and auxiliary indices for CAR, i.e., the secreted cortisol concentration within 30 min of awakening (CARscc) and absolute and relative increases in cortisol level within 30 min of awakening (CARi and CARi%, respectively). We also determined these values for auxiliary indices derived from nighttime cortisol level, i.e., the ratio of cortisol level 30 min after awakening (CA(30 min)) to nighttime level (CA(30 min)/NC), as well as absolute and relative decreases in cortisol levels from CA(30 min) to nighttime (DCd and DCd%, respectively). We found no significant differences in cortisol level for any time point or in auxiliary indices between collection days, genders and ages. CONCLUSION: The provided descriptive information and statistics on the CAR and nighttime cortisol level will be helpful to medical specialists and researchers involved in hypothalamus- pituitary-adrenal axis assessment.

Day-to-day differences in cortisol levels and molar cortisol-to-DHEA ratios among working individuals.

PURPOSE: The present study was carried out to determine day-to-day differences in cortisol levels and the molar cortisol-to-dehydroepiandrosterone (DHEA) ratio (molar C/D ratio) in working subjects. MATERIALS AND METHODS: The cortisol and DHEA levels were measured from saliva samples collected 30 minutes after awakening for 7 consecutive days in full-time working subjects that worked Monday through Saturday. To determine the day-to-day differences within subjects, the collected data was analyzed using variance (ANOVA) for a randomized complete block design (RCBD). RESULTS: The cortisol levels from samples collected 30 minutes after awakening on workdays were similar to each other, but were significantly different from the cortisol levels on Sunday. The DHEA levels were not significantly different between the days of week. The DHEA levels on Monday and Tuesday were relatively lower than the levels on the other weekdays. The DHEA levels on Thursday and Friday were relatively higher than the other days. The molar C/D ratios on Sunday were significantly lower than those on workdays. The molar C/D ratios on Monday and Tuesday were significantly higher than those on Wednesday or other workdays. CONCLUSION: The cortisol levels and the molar C/D ratios demonstrate differences in adrenocortical activities between workdays and non-workdays, but the molar C/D ratio additionally represents differences in adrenocortical status between the first two workdays and other workdays. Thus, it is possible that the day-to-day differences in the cortisol levels and the molar C/D ratio represent the adrenal response to upcoming work-related stress.

Molecular cloning and expression of steroidogenic acute regulatory protein from bullfrog (Rana catesbeiana).

Steroidogenic acute regulatory protein (StAR) transfers cholesterol from the outer mitochondrial membrane to the inner membrane where the cytochrome P450 side chain cleavage enzyme (P450scc) resides. This process is the rate-limiting step in steroidogenesis. StAR cDNAs have been cloned and characterized from a range of different species. To investigate the role of StAR in the amphibian system, we cloned a full-length StAR cDNA from bullfrog (Rana catesbeiana) using reverse transcription polymerase chain reaction (RT-PCR) in conjunction with rapid amplification of cDNA ends (RACE). The putative full-length bullfrog StAR (bfStAR) cDNA was 1862 base pairs (bp) in length, and the longest open reading frame (ORF) encoded a protein of 284 amino acids. Amino acid sequence comparison showed that amphibian StAR has a high degree of sequence identity, ranging from 62% to 98%, with StAR proteins of other species. Similar to other species, bfStAR contained two conserved domains, the mitochondrial targeting domain and cholesterol-binding domain, in the N-terminus and C-terminus of the protein, respectively. Northern blot analysis and RT-PCR indicated that StAR mRNA is expressed in the gonads and adrenal gland. Transfection of green monkey kidney (COS-1) cells with an expression construct for bfStAR revealed that it encoded 34 and 27kDa proteins that were recognized by antiserum raised against the human StAR-related lipid transfer (START) domain.

Salivary cortisol and DHEA levels in the Korean population: age-related differences, diurnal rhythm, and correlations with serum levels.

PURPOSE: The primary objective of this study was to examine the changes of basal cortisol and DHEA levels present in saliva and serum with age, and to determine the correlation coefficients of steroid concentrations between saliva and serum. The secondary objective was to obtain a standard diurnal rhythm of salivary cortisol and DHEA in the Korean population. MATERIALS AND METHODS: For the first objective, saliva and blood samples were collected between 10 and 11 AM from 359 volunteers ranging from 21 to 69 years old (167 men and 192 women). For the second objective, four saliva samples (post-awakening, 11 AM, 4 PM, and bedtime) were collected throughout a day from 78 volunteers (42 women and 36 men) ranging from 20 to 40 years old. Cortisol and DHEA levels were measured using a radioimmunoassay (RIA). RESULTS: The morning cortisol and DHEA levels, and the age- related steroid decline patterns were similar in both genders. Serum cortisol levels significantly decreased around forty years of age (p < 0.001, when compared with people in their 20s), and linear regression analysis with age showed a significant declining pattern (slope=-2.29, t=-4.297, p < 0.001). However, salivary cortisol levels did not change significantly with age, but showed a tendency towards decline (slope=-0.0078, t=-0.389, p=0.697). The relative cortisol ratio of serum to saliva was 3.4-4.5% and the ratio increased with age (slope=0.051, t=3.61, p < 0.001). DHEA levels also declined with age in saliva (slope=-0.007, t=-3.76, p < 0.001) and serum (slope=-0.197 t=-4.88, p < 0.001). In particular, DHEA levels in saliva and serum did not start to significantly decrease until ages in the 40s, but then decreased significantly further at ages in the 50s (p < 0.001, when compared with the 40s age group) and 60s (p < 0.001, when compared with the 50 age group). The relative DHEA ratio of serum to saliva was similar throughout the ages examined (slop=0.0016, t=0.344, p=0.73). On the other hand, cortisol and DHEA levels in saliva reflected well those in serum (r=0.59 and 0.86, respectively, p < 0.001). The highest salivary cortisol levels appeared just after awakening (about two fold higher than the 11 AM level), decreased throughout the day, and reached the lowest levels at bedtime (p < 0.001, when compared with PM cortisol levels). The highest salivary DHEA levels also appeared after awakening (about 1.5 fold higher than the 11 AM level) and decreased by 11 AM (p < 0.001). DHEA levels did not decrease further until bedtime (p=0.11, when compared with PM DHEA levels). CONCLUSION: This study showed that cortisol and DHEA levels change with age and that the negative slope of DHEA was steeper than that of cortisol in saliva and serum. As the cortisol and DHEA levels in saliva reflected those in serum, the measurement of steroid levels in saliva provide a useful and practical tool to evaluate adrenal functions, which are essential for clinical diagnosis.