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Background: The adrenocortical system and copeptin as prognostic markers were intensively investigated in critical illness. The potential predictive power of apelin-13 as a biomarker is largely unknown. We aimed to investigate the prognostic role of apelin-13 in relation to free cortisol, aldosterone, CRH, and copeptin in critically ill patients. Methods: In this prospective observational study, 124 critically ill patients (64 men, 60 women, median age: 70 (59–78) years) were consecutively enrolled at the time of admission. All routinely available clinical and laboratory parameters were evaluated and correlated to hormonal changes. Results: Serum apelin-13 was 1161 (617–2967) pg/mL in non-survivors vs. 2477 (800–3531) pg/mL in survivors (p = 0.054). The concentrations of apelin-13 and CRH had strong positive correlations (r = 0.685, p < 0.001) and were significantly higher in surviving non-septic patients (Apelin-13 (pg/mL): 2286 (790–3330) vs. 818 (574–2732) p < 0.05; CRH (pg/mL) 201 (84–317) vs. 89 (74–233) p < 0.05). Apelin-13 and free cortisol were independent determinants of survival in the multivariate Cox regression analysis, while copeptin, CRH, or aldosterone were not. Conclusions: Beyond free cortisol, serum apelin-13 may also help refine prognostic predictions in the early phase of critical illness, especially in non-septic patients. [ABSTRACT FROM AUTHOR]

Serum cortisol mainly binds to the cortisol-binding globulin (CBG). Children with biliary atresia (BA) may have low serum CBG levels; thus, low serum total cortisol (TC) levels and adrenal insufficiency (AI) may be overdiagnosed. This study aimed to assess adrenal function in children with BA. All the patients underwent adrenocorticotropic hormone (ACTH) stimulation tests. Plasma ACTH, serum TC, and CBG levels were measured at baseline, with additional TC measurements at 30 and 60 min during testing. Free cortisol (FC) index (FCI) and calculated FC (cFC) were also calculated. AI was defined as peak TC <500 nmol/L (<18 µg/dL), peak FCI <12 nmol/mg, or peak cFC <33 nmol/L (<1.2 µg/dL). This study enrolled 71 children with BA. The Median (IQR) age of the patients was 5.5 (1.7-11.4) years. Twenty-five (35%) patients were diagnosed with AI based on the peak TC. In the AI group, the median serum CBG level was significantly lower than that in the non-AI group (481 vs. 533 nmol/L, p = 0.03). Only eight patients (11%) met all three AI criteria (six secondary AI and two primary AI). In conclusion, low serum CBG levels contribute to reduced peak TC and, consequently, overdiagnosing AI. Peak FCI and cFC could help reduce the overdiagnosis of AI. [ABSTRACT FROM AUTHOR]

Chronic caloric deprivation and obesity are complicated by hypercortisolemia. The effects of acute overfeeding and fasting on circulating free cortisol levels and conversion of cortisone to free cortisol are unknown. We hypothesized that serum-free cortisol and free cortisol-to-cortisone ratio would increase after both overfeeding and fasting. This is a prospective study of 22 healthy volunteers who completed a 10-day high-calorie protocol followed by a 10-day fast, separated by a 2-wk washout. Morning free and total cortisol and free cortisone levels (LC/MS) were measured at baseline and after 10 days of each intervention. Both highcalorie feeding and fasting increased total and free cortisol and the free cortisol-to-free cortisone ratio (P = 0.001 to P = 0.046). There were sex interactions, with significant effects in men (P < 0.001), but not in women (P = 0.898 and 1.000, respectively) in subset analyses examining the effects of fasting on free cortisol and the free-to-total cortisol ratio. Overfeeding and fasting both increase circulating free cortisol levels and appear to alter the balance between cortisol and its inactive metabolite, cortisone. Further study is warranted to determine whether elevated cortisol levels contribute to complications of starvation and obesity, such as bone fragility. NEW & NOTEWORTHY Overfeeding and fasting both increase circulating free cortisol levels and appear to alter the balance between cortisol and its inactive metabolite, cortisone. The effect of fasting on free cortisol levels is modified by sex. Further study is needed to determine the mechanisms driving the increases in cortisol. [ABSTRACT FROM AUTHOR]

Purpose: Cortisol plays a role in the regulation of metabolic homeostasis. Dexamethasone is a powerful synthetic glucocorticoid that acts as an anti-inflammatory and immunosuppressive agent. Plasma free cortisol, which has a low concentration in the blood, is the cortisol fraction that provides biological activity. It is emphasized that measurement of dexamethasone, total and free cortisol levels can directly affect the results of dexamethasone suppression test, which is routinely used to exclude endogenous hypercortisolemia in patients with adrenal adenoma, and is important in differential diagnosis. In this study, a precise and rapid method was developed for the measurement of dexamethasone, total and free cortisol levels in plasma samples, besides demonstrating the clinical applicability of the method with samples from adrenal adenoma patients. Material and Methods: Method optimization studies were performed using liquid chromatographytandem mass spectrometry with stable isotope dilution-multiple reaction monitoring. Results: The assay demonstrated a good linear dynamic range of 0.5-20 µg/L, 5-200 µg/L and 0.5-100 µg/L for dexamethasone, total and free cortisol, respectively. The values for intra- and inter-day precisions of analytes were =6.9% with the accuracies ranging from 91.6% to 113.0%. Conclusion: Measurement of these parameters can be used reliably to diagnose diseases causing hypercortisolemia. [ABSTRACT FROM AUTHOR]

Prenatal exposure to heavy metals is known to be associated with adverse birth outcomes and oxidative stress biomarkers. In this study, we examined whether maternal free cortisol or 8-Hydroxy-2-Deoxyguanosine (8-OHdG) could mediate associations between maternal heavy metal exposure and birth outcomes. A total of 182 healthy pregnant women were recruited. Heavy metals (including Pb, Hg, and Cd), free-cortisol, and 8-OHdG were analyzed in urine at delivery. Birth outcomes including birth weight, length, Ponderal index, and head circumference were measured. To examine associations of maternal urinary heavy metals with biomarkers and birth outcomes, generalized linear models were employed. Birth length was positively associated with Pb (β = 0.78, 95% CI: 0.09–1.46) and Hg (β = 0.84, 95% CI: 0.23–1.45) (both p < 0.05). The Ponderal index, a measure of a newborn's leanness, was negatively associated with maternal urinary Pb (β = −0.23, 95% CI: −0.46–−0.07) and Hg (β = −0.26, 95% CI: −0.44–−0.08) (both p < 0.05). No association between maternal Cd and birth outcomes was observed. Most heavy metals showed positive associations with free cortisol and 8-OHdG. Free cortisol was identified as a mediator underlying the observed relationship between Hg and birth length or Ponderal index. This study observed adverse birth outcomes from maternal exposures to Pb and Hg. Increased free cortisol related to Hg exposure was suggested as a possible causal pathway from Hg exposure to birth outcomes such as the Ponderal index. [ABSTRACT FROM AUTHOR]

Recurrent aphthous stomatitis (RAS) is a commonly-encountered oral lesion. The aim of this study was to identify the levels of stress, anxiety and depression in patients with RAS .Fourty (40) patients with RAS and 40 age-matched controls were enrolled in this study. Blood and salivary levels were analyzed electrochemiluminescence technique. The Beck Depression Rating Scale (BDRS) and Beck Anxiety Scale (BAS), Perceived Stress Scale (PSS), and State-Trait Anxiety Inventory (STAI-S and STAI-T) were applied to all participants. There was no statistically significant differences levels between two groups regarding to BDRS, BAS, PSS, STAI-S and STAI-T level. Blood cortisol levels were statistically significantly higher in patients with RAS than controls (12.45 ± 0.74 µg/dL vs. 9.8± 0.68,respectively,p=0.01), wheras there was no statistically significant difference between women with RAS than controls regarding to salivary cortisol levels (0.33± 0.03 µg/dL vs. 0.34± 0.03, respectively. Patients with RAS have not higher depression and anxiety levels compared to the control. [Med-Science 2020; 9(1.000): 170-4]

Context: Some patients with classic congenital adrenal hyperplasia (CAH) survive without glucocorticoid treatment. Increased precursor concentrations in these patients might lead to higher free (biological active) cortisol concentrations by influencing the cortisol-protein binding. In 21-hydroxylase deficiency (21OHD), the most common CAH form, accumulated 21-deoxycortisol (21DF) may further increase glucocorticoid activity. Both mechanisms could explain the low occurrence of symptoms in some untreated classic CAH patients., Objective: Develop and validate an LC-MS/MS method for free cortisol and free 21DF to quantify these steroids in untreated patients with classic CAH (n=29), non-classic CAH (NCCAH, n=5), other forms of adrenal insufficiency (AI, n=3), and controls (n=11) before and 60 minutes after Synacthen® administration., Results: Unstimulated total cortisol concentrations of untreated classic CAH patients (median 109 nmol/L) were lower than in untreated NCCAH patients (249 nmol/L, p=0.010) and controls (202 nmol/L, p=0.016), but free cortisol concentrations were similar. Basal free 21DF concentrations were high in 21OHD patients (median 5.32 nmol/L) and undetectable in AI patients and controls (<0.19 nmol/L). After Synacthen® administration, free 21DF concentrations increased in 21OHD patients, while free cortisol concentrations did not change., Conclusions: Free cortisol concentrations in classic CAH patients were similar to those in controls and NCCAH patients, indicating comparable cortisol availability. Additionally, 21OHD patients produce high concentrations of 21DF, possibly explaining the low occurrence of symptoms in some classic 21OHD patients. Free cortisol and 21DF levels should be considered in evaluating adrenal insufficiency in patients with CAH., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society.)

Autonomous cortisol secretion (ACS) of an adrenal incidentaloma (AI) is associated with mild cortisol excess that could result in poor metabolic and cardiovascular outcomes. The biological activity of glucocorticoids depends on the unbound, free fraction. We aimed to evaluate plasma free cortisol (FC) concentrations in patients with ACS in this cross-sectional study. One hundred and ten AI patients in 3 groups; non-functioning (NFA, n=33), possible ACS (n=65), ACS (n=12) were enrolled. Following measurements were conducted: Clinical data and total serum cortisol (TC), plasma corticotrophin (ACTH), serum dehydroepiandrosterone sulfate (DHEA-S), cortisol after 1 mg dexamethasone by both immunoassay and LC-MS/MS (DexF), serum corticosteroid binding globulin (CBG), plasma dexamethasone concentration [DEX] and plasma FC by LC-MS/MS. Patients with ACS featured an unfavorable metabolic profile. Plasma [DEX] and serum CBG levels were similar between groups. Plasma FC was significantly higher in ACS when compared to NFA and possible ACS groups p<0.05 and p<0.01, respectively. In multiple regression analysis DexF (beta=0.402, p<0.001) and CBG (beta=−0.257, p=0.03) remained as the independent predictors of plasma FC while age, sex, BMI, smoking habit, and existing cardiovascular disease did not make a significant contribution to the regression model. In conclusion, the magnitude of cortisol excess in ACS could lead to increased plasma FC concentrations. Further studies in AI patients are needed to demonstrate whether any alterations of cortisol affinity for CBG exist and to establish whether plasma FC concentrations predict the unfavorable metabolic profile in ACS. [ABSTRACT FROM AUTHOR]

Prenatal exposure to heavy metals is known to be associated with adverse birth outcomes and oxidative stress biomarkers. In this study, we examined whether maternal free cortisol or 8-Hydroxy-2-Deoxyguanosine (8-OHdG) could mediate associations between maternal heavy metal exposure and birth outcomes. A total of 182 healthy pregnant women were recruited. Heavy metals (including Pb, Hg, and Cd), free-cortisol, and 8-OHdG were analyzed in urine at delivery. Birth outcomes including birth weight, length, Ponderal index, and head circumference were measured. To examine associations of maternal urinary heavy metals with biomarkers and birth outcomes, generalized linear models were employed. Birth length was positively associated with Pb (β = 0.78, 95% CI: 0.09–1.46) and Hg (β = 0.84, 95% CI: 0.23–1.45) (both p < 0.05). The Ponderal index, a measure of a newborn’s leanness, was negatively associated with maternal urinary Pb (β = −0.23, 95% CI: −0.46–−0.07) and Hg (β = −0.26, 95% CI: −0.44–−0.08) (both p < 0.05). No association between maternal Cd and birth outcomes was observed. Most heavy metals showed positive associations with free cortisol and 8-OHdG. Free cortisol was identified as a mediator underlying the observed relationship between Hg and birth length or Ponderal index. This study observed adverse birth outcomes from maternal exposures to Pb and Hg. Increased free cortisol related to Hg exposure was suggested as a possible causal pathway from Hg exposure to birth outcomes such as the Ponderal index.

Background: The aim of the study was to examine the relationship between serum total cortisol (TC) and free cortisol (FC) levels in children with septic shock and the relationship of these levels with baseline illness severity. Methods: A sub-study of a randomized controlled trial (RCT) of hydrocortisone vs. placebo in pediatric septic shock conducted in seven academic pediatric intensive care units (PICUs) in Canada on children aged newborn to 17 years. Thirty children with septic shock had serum sent for TC and FC measurement within 6 h of meeting the study eligibility criteria. Results: Baseline FC and TC levels were strongly correlated with baseline Pediatric Risk of Mortality (PRISM) score (R2 = 0.759, p < 0.001; R2 = 0.717, p < 0.001) and moderately correlated with admission Vasotropic Inotropic Score (VIS) (R2 = 0.489, p < 0.001; R2 = 0.316, p < 0.001). Serum TC levels were highly correlated with FC levels (R2 = 0.92, p < 0.001) and showed strong agreement (R2 = 0.98, p < 0.001 on a Bland-Altman plot). The ratio of FC to TC moderately correlated with TC levels (R2 = 0.46, p < 0.001) but did not correlate with baseline albumin levels (R2 = 0.19, p = 0.13). Conclusions: Random TC and FC levels are strongly correlated, show strong agreement and are reflective of illness severity in children with septic shock. As such, isolated FC measurement does not appear to provide added information relative to TC in acutely ill children with septic shock. [ABSTRACT FROM AUTHOR]

Several studies suggested potential links of phthalates to stress-related outcomes. However, limited evidence has been available for the relationships between phthalate metabolites and free cortisol and 8-hydroxy-2′-deoxyguanosine (8-OHdG) in perinatal and postnatal environments. Therefore, we evaluated the relationships between phthalate metabolites and free cortisol and 8-OHdG in mother–child pairs. We repeatedly collected urine samples of 287 mother–child pairs from just before delivery to 15 months of age to measure the levels of four phthalate metabolites – mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono-isobutyl phthalate (MiBP), and mono- n -butyl phthalate (MnBP) – and free cortisol and 8-OHdG. We used linear mixed effect models and generalized additive mixed models to estimate the relationship between the phthalate metabolites and free cortisol and 8-OHdG after adjusting for the child's gender, urine collection time, and maternal smoking status. The four phthalate metabolite levels were strongly correlated each other (all, p  < .0001), and intra-class correlation for each metabolite in children ranged from 0.18 to 0.96. All four phthalate metabolites were positively associated with both free cortisol (MEHHP, β = 0.18 and p  < .0001; MEOHP, β = 0.17 and p  < .0001; MiBP, β = 0.13 and p  = .0001; MnBP, β = 0.21 and p  < .0001; and molar sum of metabolites, β = 0.21 and p  < .0001) and 8-OHdG (MEHHP, β = 0.20 and p  < .0001; MEOHP, β = 0.18 and p  < .0001; MiBP, β = 0.23 and p  < .0001; MnBP, β = 0.28 and p  < .0001; and molar sum of metabolites, β = 0.29 and p  < .0001) in childhood. Our findings suggest that phthalate exposures increase free cortisol and 8-OHdG levels in early childhood. [ABSTRACT FROM AUTHOR]

Pregnancy is one of the defining characteristics of placental mammals. Key in the growth and development of the fetus during pregnancy are the dynamics of glucocorticoids (GCs) and their binding protein,corticosteroid-binding globulin (CBG), which determines how much of the GCs are free and biologically active. Out of more than 5000 species of placental mammals in 19 different orders, our understanding of the dynamics of maternal GCs and CBG during pregnancy is largely limited to the detailed study of 3 groups – sheep, laboratory rodents, and humans. The assumption is often made that what we see in these few species applies to the rest. To examine this generality, we compared patterns of maternal GCs over pregnancy from all placental mammals where data is available: in the blood of 13 species from 5 different orders and in metabolites in excreta in an additional 20 species from 9 orders. We found that maternal free GCs increase by late pregnancy in most taxa. This increase is achieved by either an increase in total GC secretion or a decrease in CBG. A major exception is found in the even-toed ungulates (sheep, cows, etc.) where maternal GCs and CBG remain stable, but where the fetal adrenals mature in late pregnancy and produce the majority of their own GCs. We conclude that patterns of change in maternal GCs and CBG during pregnancy are species-specific but are alternative means to the same end: increased fetal exposure to GCs in late pregnancy, which is essential for development. [ABSTRACT FROM AUTHOR]

Cortisol levels reflect hypothalamic-pituitary-adrenocortical (HPA) axis activity. While most plasma cortisol is supposed to be bound to the soluble corticosteroid-binding globulin (CBG), only free cortisol (FC) actively regulates metabolic and immunological processes. We aimed to establish a multispecies suitable method to assess FC in cows and horses which in combination with total cortisol (TC) allows interpreting proportional changes of cortisol in saliva as well as in blood in response to a standardized HPA axis activation via ACTH. We further investigated if the ratios of cortisol fractions as obtained at basal levels in healthy horses (herbivorous and monogastric) and dairy cows (herbivorous and ruminant) change during HPA axis activation, and to which extent saliva cortisol (SC) is representative for alterations in plasma FC and adrenal cortex reactivity. However, it was not the objective of the present study to directly compare the two species. Dosages of ACTH applied in cows and horses were based on published data. Synthetic ACTH was intravenously administered to eight dairy cows (0.16 µg/kg BW) and five horses (1 μg/kg BW). Blood and saliva were collected every 30 min for 3 h from a jugular vein catheter, and analyzed for TC and SC, the ratio of free cortisol (rFC), and the concentration of FC (cFC) in plasma. During the entire sampling period of the ACTH test, plasma TC was paralleled by blood cFC, rFC, and SC in both cows and horses. All cortisol fractions increased within 30 min of ACTH administration compared to basal values (0 min, P < 0.05). Peak TC concentration reached 63.2 ± 9.6 ng/mL and 73.2 ± 11.8 ng/mL in bovine and equine plasma, respectively. Peak values of rFC averaged 17.9 ± 4.5% in cows and 19.2 ± 7.8% in horses. The ratio of SC to cFC in horses remained similar during the ACTH challenge suggesting that SC is recruited from plasma FC. However, SC increased less compared to plasma TC and FC during HPA axis activation in cows. In conclusion, the short-term activation of the HPA axis caused not only an elevation of TC, but also a similar increase of rFC in both species. SC closely reflected changes of FC in horses, but less accurately in cows. The concomitant evaluation of changes among cortisol fractions might give further indications on adaptation mechanisms in glucocorticoid regulation as well as differentiate cortisol-related health disorders. [ABSTRACT FROM AUTHOR]

Male reproductive success in most mammals is determined by their success in direct inter-male competition through aggression and conflict, resulting in female-defense mating systems being predominant. This is linked to male testosterone levels and its dynamics. However, in certain environments, a scramble-competition mating system has evolved, where female reproductive behavior takes precedence and male testosterone dynamics are unlikely to be related to inter-male competition. We studied the North American red squirrel ( Tamiasciurus hudsonicus ), a species with a well-established scramble-competition system. Using an ACTH hormonal challenge protocol as a proxy for competitive interactions, we compared the testosterone dynamics in breeding males in late winter with that in nonbreeding males in late spring in the Yukon. To gain an integrated picture of their physiological state, we also assessed changes in their stress response, body mass, energy mobilization, and indices of immune function. Testosterone levels at the base bleed were high in breeding males (2.72 ng/mL) and virtually absent in non-breeding males (0.04 ng/mL). Breeding males were in better condition (heavier body mass, higher hematocrit, and higher erythrocytes), had higher indices of immune function (neutrophil:lymphocyte ratio), but a similar ability to mobilize energy (glucose) compared with non-breeding males. Though total cortisol was higher in non-breeding males, free cortisol was twice as high in breeding males as their corticosteroid binding globulin levels were half as high. In response to the ACTH challenge, testosterone levels in breeding males declined 49% over the first hour and increased 36% over the next hour; in non-breeding males levels showed no change. Free cortisol increased only modestly (26% in breeding males; 23% in non-breeding males). Glucose levels changed similarly in breeding and nonbreeding males, declining for the first 30 min and then increasing for the next 60 min. Thus, testosterone and components of the stress axis function in a profoundly different manner in male red squirrels than in males of mammals with female-defense mating systems. There are four probable interrelated reasons for these adaptations in male red squirrels: the marginal benefits of each mating, the constraints of mate searching away from their own resource-based territories, energy mobilization in a harsh environment, and a long life span. [ABSTRACT FROM AUTHOR]

Purpose: The appropriate cosyntropin dose during cosyntropin stimulation tests remains uncertain. We conducted a prospective, randomized pilot study to compare 1 μg IV low dose cosyntropin test, 25 μg IM medium dose cosyntropin test, and 250 μg IM standard dose cosyntropin test to evaluate secondary adrenal insufficiency. Insulin tolerance test was used as the gold standard. Method: The study included patients with hypothalamic/pituitary disease ( n = 10) with at least one pituitary axis deficiency other than ACTH deficiency and controls ( n = 12). All tests were done in random order. Sensitivity and specificity were calculated for total cortisol and serum free cortisol cut-off levels during cosyntropin stimulation tests. Results: The median (range) age and F/M sex ratios for patients and controls were 54 years (23-62), 2/8, and 33 years (21-51), 6/6, respectively. The best total cortisol cut-off during low dose cosyntropin test, medium dose cosyntropin test, 30 min and 60 min standard dose cosyntropin test were 14.6 μg/dL (100% sensitivity & specificity), 18.7 μg/dL (100% sensitivity, 88% specificity), 16.1 (100% sensitivity & specificity), and 19.5 μg/dL (100% sensitivity & specificity), respectively. There was no difference in the ROC curve for cortisol values between the cosyntropin stimulation tests ( p > 0.41). Using a cortisol cut-off of 18 μg/dL during cosyntropin stimulation tests, only cortisol level at 30 min during standard dose cosyntropin test provided discrimination similar to insulin tolerance test. The best peak free cortisol cut-off levels were 1 μg/dL for insulin tolerance test, 0.9 μg/dL for low dose cosyntropin test, 0.9 μg/dL for medium dose cosyntropin test, and 0.9 μg/dL and 1.3 μg/dL for 30 min and 60 min standard dose cosyntropin test, respectively. Conclusion: All cosyntropin stimulation tests had excellent correlations with insulin tolerance test, when appropriate cut-offs were used. This pilot study does not suggest an advantage in using 25 μg cosyntropin dose during the cosyntropin stimulation test. A serum free cortisol cut-off of 0.9 μg/dL may be used as pass criterion during low dose cosyntropin test, standard dose cosyntropin test cosyntropin test, and 30 min standard dose cosyntropin test. [ABSTRACT FROM AUTHOR]

This study aimed to examine the effects of several essential oils on insomnia in dementia patients following transdermal treatment (aromatherapy). The mean change rates (%) of sleep biomarkers were compared between the single essential and jojoba (vehicle) oil massage groups in this study. The lavender (L) essential oil massage group demonstrated a significant decrease in the mean change rate (%) of 24-h urinary free cortisol, whereas the valerian (V) essential oil massage group demonstrated a significant increase in the mean change rate (%) of serum 5-hydroxytryptamine. In addition, a significant increase in the mean change rate (%) of 24-h urinary norepinephrine was observed in the chamomile (C) essential oil massage group only. Based on these results, valerian, lavender, and chamomile oils were mixed in different ratios to produce blending oils A (L:C:V=2:2:1), B (L:C:V=3:1:1) and C (L:C:V=1:3:1). The highest level of serum 5-hydroxytryptamine was observed after administering blending oil A. These results suggest that blending oil A might possess therapeutic effects against insomnia. Overall, it is hypothesized that the optimally blended essential oil will produce synergic effects when combined with hypnotic drugs.

Five musicians suffering from focal dystonia participated in a pilot study that examined the feasibility of an experimental protocol designed to assess musicians' motor performance under stress. Electrocardiography, free cortisol levels, and subjective assessments were used to monitor alterations of the hypothalamic-pituitary-adrenal axis. As measures of motor outcome, temporal variability of finger movements and muscular cocontraction of the wrist flexor and extensor were assessed. Findings suggest that the specific experimental design could be successfully applied. Several methodological issues such as carryover effects, the use of free cortisol, the inclusion of a double baseline, and the classification of dystonic patients into stress responders and nonresponders are analyzed and discussed. [ABSTRACT FROM AUTHOR]

Background: Normative data have been established for stimulated serum total cortisol in children but not for serum free cortisol. Methods: Children who were referred for ACTH testing to rule out adrenal insufficiency were enrolled. Only children with normal response and normal androgen levels were included. Total cortisol was determined by a chemiluminescence method, and free cortisol was measured by the same method following equilibrium dialysis. Results: The study group consisted of 85 subjects (28 male; 57 female) with a median age of 8.5 years (range 0.6-17.7). The mean basal and peak total cortisol levels were 11.5 ± 5.7 and 32.9 ± 6.2 μg/dl, respectively. The mean basal and peak free cortisol levels were 0.4 ± 0.3 and 1.8 ± 0.6 μg/dl, respectively. There was a negative correlation between peak total cortisol and age but not between peak free cortisol and age. The 3rd and 97th percentile values for peak free cortisol were 0.94 μg/dl (26 nmol/l) and 2.97 μg/dl (82 nmol/l), respectively. Conclusions: Measurement of free cortisol has the advantage of being independent of cortisol-binding globulin levels. This study provides reference ranges for stimulated free cortisol in children, with a cutoff value of 0.9 μg/dl (25 nmol/l) as a normal response to a standard ACTH test. [ABSTRACT FROM AUTHOR]

Pregnancy and lactation are key times in the life of female mammals when energetic resources must be brought to bear to produce and nurture offspring. Changes in glucocorticoid (GC) levels are central to this objective, due to their roles in modulating development and physiology and in mediating energetic tradeoffs. We examined GC changes over reproduction in a species living in a harsh seasonal environment: the arctic ground squirrel ( Urocitellus parryii ). Females become pregnant immediately after emerging from a ∼9 month hibernation, and then must begin this pregnancy during an additional month of freezing temperatures and limited food availability. We measured plasma levels of total cortisol, corticosteroid-binding globulin (CBG), and free cortisol in unstressed females at three stages: not-visibly pregnant, visibly pregnant, and lactating. Total cortisol levels were similar in all stages, but CBG levels increased 4-fold from the not-visibly pregnant stage to visibly pregnant and lactating stage. As a result, the free cortisol fraction declined from 51% of total cortisol when females were not-visibly pregnant to only 5% when they were visibly pregnant (remaining low and stable throughout pregnancy) and 10% when they were lactating. This pattern is markedly different from that seen in other mammals, where 10% or less of GCs are free and these tend to increase during gestation. We postulate that the high free cortisol just prior to visible pregnancy is a seasonal adaptation relating either to the pronounced physiological changes the female must undergo after emerging from hibernation and immediately getting pregnant, or to the mobilization of body reserves for energy to permit pregnancy, or both. Thereafter, high CBG levels may shield the developing offspring from the negative effects of cortisol overexposure. [ABSTRACT FROM AUTHOR]

Objectives To investigate the agreement between the calculated free cortisol levels according to widely applied Coolens and adjusted Södergård equations with measured levels in the critically ill. Design and methods A prospective study in a mixed intensive care unit. We consecutively included 103 patients with treatment-insensitive hypotension in whom an adrenocorticotropic hormone (ACTH) test (250 μg) was performed. Serum total and free cortisol (equilibrium dialysis), corticosteroid-binding globulin and albumin were assessed. Free cortisol was estimated by the Coolens method (C) and two adjusted Södergård (S1 and S2) equations. Bland Altman plots were made. Results The bias for absolute (t = 0, 30 and 60 min after ACTH injection) cortisol levels was 38, − 24, 41 nmol/L when the C, S1 and S2 equations were used, with 95% limits of agreement between − 65–142, − 182–135, and − 57–139 nmol/L and percentage errors of 66, 85, and 64%, respectively. Bias for delta (peak-baseline) cortisol was 14, − 31 and 16 nmol/L, with 95% limits of agreement between − 80–108, − 157–95, and − 74–105 nmol/L, and percentage errors of 107, 114, and 100% for C, S1 and S2 equations, respectively. Conclusions Calculated free cortisol levels have too high bias and imprecision to allow for acceptable use in the critically ill. [ABSTRACT FROM AUTHOR]

Corticosteroid-binding globulin (CBG) is the principal transport protein of glucocorticoids. Approximately 80–90% of serum cortisol binds to CBG with high affinity and only about 5% of cortisol remain unbound and is considered biologically active. CBG seems to modulate and influence the bioavailability of cortisol to local tissues. In this review, we will discuss physicochemical properties of CBG and structure of CBG in the mechanisms of binding and release of cortisol. This review describes several factors affecting CBG functions, such as genetic factors or temperature. Furthermore, clinical implications of CBG abnormalities and the measurement of CBG and its use for assessment of free cortisol levels are described in this review. [ABSTRACT FROM AUTHOR]

Background: Dissociation between plasma-free cortisol and total cortisol profiles exists in critical illness. Data on plasma-free cortisol are based on either calculated values or immunoassay-based measurements. Both have significant limitations. Statins have been advocated as a therapy in sepsis. Whether they impact on plasma cortisol through inhibition of cholesterol synthesis is unclear. Objectives: In patients enrolled into a randomized trial of statins in sepsis (n = 250), we examined the association of mass spectrometry measured plasma-free cortisol, plasma-free cortisol/plasma total cortisol ratios, and outcome and the impact of concomitant statin therapy on cortisol profiles in 80 steroid naïve patients. Patients: Two hundred twenty serial measurements of plasmafree cortisol, plasma total cortisol, and interleukin-6 were collected from 80 patients (43 placebo and 37 statins). Data from 10 volunteers were used as controls. Measurements and Main Results: Data are presented as median and interquartile range. Compared with controls, in severe sepsis, baseline plasma total cortisol was elevated two-fold (463 nmol/L [284–742 nmol/L] vs 245 nmol/L [200–299 nmol/L], p < 0.001), plasma-free cortisol 20-fold (75 nmol/L [20–151 nmol/L] vs 5 nmol/L [5–7 nmol/L], p < 0.001), and plasma-free cortisol/plasma total cortisol ratio six-fold (0.15 vs 0.02, p = 0.058). Baseline interleukin-6 was elevated at 121 pg/mL (65–611 pg/mL). In severe sepsis, there were no differences in plasma total cortisol (p = 0.66), plasma-free cortisol (p = 0.77), and interleukin-6 (p = 0.29) between statins and placebo groups. Plasma-free cortisol, plasma total cortisol, and plasma-free cortisol/plasma total cortisol were positively correlated with interleukin-6 (p = 0.0001, p < 0.0004, and p < 0.001, respectively) and day 90 mortality (p = 0.03, p = 0.03, and p = 0.058, respectively). Elevated plasma-free cortisol/plasma total cortisol ratios were associated with increased length of stay (p = 0.04). Baseline plasma-free cortisol, plasma total cortisol, and plasma-free cortisol/plasma total cortisol ratios were higher in nonsurvivors as compared with survivors (174 nmol/L [77–329 nmol/L] vs 57 nmol/L [17–122 nmol/L], p = 0.016; 890 nmol/L [333–1,430 nmol/L] vs 408 nmol/L [269–681 nmol/L], p = 0.035; and 0.19 [0.13–0.29] vs 0.14 [0.07–0.20]; p = 0.054, respectively). Conclusions: In severe sepsis, plasma-free cortisol increase is 10-fold greater than that of plasma total cortisol. Both are similarly associated with inflammatory response and mortality. Elevated plasma-free cortisol/ plasma total cortisol ratios were associated with increased length of stay. Statin therapy does not influence the plasma cortisol profiles in patients with severe sepsis. [ABSTRACT FROM AUTHOR]

Abstract: Background: Insulin-induced hypoglycemia (IHT) is considered the gold standard test for evaluating the HPA axis. Serum free cortisol or its surrogate, salivary cortisol as opposed to total cortisol concentrations, offers a better reflection of the activation of HPA axis. Our study aimed to derive reference ranges for the normal salivary cortisol levels in healthy patients and patients with adrenal insufficiency. Design and methods: Serum cortisol concentrations, using the gold standard of IHT, and salivary cortisol were obtained. 36 patients referred to our outpatient endocrine testing unit for evaluation of adrenal function were included in the study. Most subjects had a history of suspected hypothalamic/pituitary disease causing adrenal insufficiency. Results: We found a strong linear correlation between the serum and salivary cortisol concentrations in simultaneously collected samples (r=0.81, 95% CI 0.74–0.86, p<0.0001). The corresponding salivary cortisol equivalent to a serum cortisol of 500nmol/L, using a linear-regression equation, was 16.7nmol/L (95% CI 13.3–20.1nmol/L, p=0.0001). A salivary cortisol of 13.3nmol/L has a specificity of 89.3% to detect abnormal HPA function. Using the upper 95% CI result of salivary cortisol 20.1 yields a sensitivity of 87.5%. Conclusion: With the present assay, adrenal insufficiency may be diagnosed with reasonable confidence if a random salivary cortisol is lower than 13.3nmol/L and excluded if a random salivary cortisol is higher than 20.1nmol/L. Future studies should correlate these thresholds with clinical outcomes. [Copyright &y& Elsevier]

The free hormone hypothesis states that a hormone's physiological effects depend on the free hormone concentration, not the total hormone concentration. Although the in vivo relationship between free hormone and protein-bound hormone is complex, most experts have applied this view to the design of assays used to assess the free hormone concentration in the blood sampled for testing in vitro. The history of the measurement of free thyroxine, probably the most frequently requested free hormone determination, offers a good example of the approaches that have been taken. Methods that require physical separation of the free hormone from the protein-bound hormone must address both the potential disturbance in the equilibrium between the two, as well as the challenge of quantifying small levels of hormone accurately and precisely. The implementation of mass spectrometry in the clinical laboratory has helped to develop proposed reference measurement procedures. These must be utilized to standardize the variety of immunoassay approaches that currently represent options commercially available to the routine clinical laboratory. Practicing endocrinologists should discuss the details of the free hormone assays offered by the clinical laboratory they utilize for patient result reporting, and clinical laboratories should implement the recommendations of published guidelines to ensure that free hormone results using commercially available immunoassays are as accurate and precise as possible. [Copyright &y& Elsevier]

Abstract: The predominant carrier of cortisol in circulation is corticosteroid-binding globulin (CBG) which is a non-functional member of the family of serine protease inhibitors. Corticosteroid-binding globulin possesses an exposed elastase sensitive loop and upon cleavage it adopts a “relaxed” conformation promoting the delivery of cortisol to sites of inflammation. Recently we have developed monoclonal antibodies which recognise only the intact exposed elastase loop, including an N-glycosylation site, which, in concert with another monoclonal antibody to CBG, offered the potential for the determination of intact and total CBG which may both be present in circulation. Here we validate these parallel ELISAs and show that like total CBG there is little diurnal variation of intact plasma CBG. Furthermore in a normal reference population the majority of CBG is in the intact or active form but a significant level of apparently cleaved CBG is evident. In some subjects there is gross discordance between total CBG and intact CBG implying a predominance of apparently cleaved CBG in circulation and this significantly affects calculated free cortisol levels. Gross differences in total and intact CBG levels may not be due to differences in N-glycosylation affecting antibody binding as CBG levels are unaffected by PNGase F treatment. [Copyright &y& Elsevier]

Live-capture is a necessary component for the scientific study and management of most mammals, but it may negatively affect their health and physiology. We compared blood parameters related to the stress response (nominal base levels) from red squirrels Tamiasciurus hudsonicus after capture of up to 4.5 h in five different live trap models ( Hava-hart, Sherman, Tomahawk 102, Tomahawk 103 and ' Special Squirrel' trap) with true base levels (obtained in less than three minutes). In addition, we evaluated the capture rate in the five trap models. We found that (1) prolonged time in live traps altered stress hormone concentrations compared with true base levels, but maximum corticosteroid-binding capacity was unaffected; (2) squirrels captured in a trap model with reduced visibility (a roof cover - Hava-hart) had significantly lower ( c. 50%) mean free cortisol levels compared with those captured in a trap model with full visibility ( Tomahawk 102), but all other blood parameters were similar; (3) cortisol levels and white blood cell counts (mainly neutrophil counts) were positively related to duration of capture; (4) a non-covered trap ( Tomahawk 102) was most effective and fully covered trap ( Sherman) was least effective at capturing squirrels. We discuss the use of effective, yet less stress-inducing trap models to mitigate the stress caused by live-capture on these animals. We conclude that covered traps such as the Hava-hart may reduce trap-induced stress in red squirrels, but at the same time also reduces their capture rates. [ABSTRACT FROM AUTHOR]

Abstract: Corticosteroid-binding globulin (CBG) is the principal carrier of cortisol in circulation and is a non-inhibitory member of the serpin family of serine proteinase inhibitors. It possesses an exposed elastase specific site which, when cleaved, allows a conformational change promoting the delivery of cortisol to sites of inflammation. Previously there was no ability to independently distinguish between the uncleaved, stressed, conformer of CBG and total CBG in circulation. Here we raised and characterized monoclonal antibodies generated against a synthetic peptide spanning the elastase cleavage site within the exposed reactive centre loop (RCL) and measured changes in CBG by ELISA following treatment with human neutrophil elastase. The antibodies recognized the synthetic peptide as well as intact CBG and the epitope (STGVTLNL) spanned the elastase cleavage site. Treatment of plasma with elastase resulted in a complete loss of CBG levels determined using these RCL antibodies whereas CBG levels measured with an unrelated CBG monoclonal antibody were unaffected. We also compared plasma levels of CBG measured by RCL antibodies and an unrelated CBG antibody and showed discordance in some samples. This study shows for the first time the ability to measure the intact, stressed conformer of CBG. We report discordance with total CBG in some samples implying the presence of cleaved CBG in circulation. This is an important finding as it has implications for free cortisol which hitherto have been determined from total cortisol and total CBG levels. This antibody could be used for determining the time course of intact CBG in various relevant patient cohorts and for structure/function studies on the biology of human CBG. [Copyright &y& Elsevier]

A central feature of the endocrine pathophysiology of septic shock is thought to be the existence of adrenal dysfunction. Based on changes in glucocorticoid secretion and responsiveness, protein binding, and activity. These changes have been described by the terms “Relative Adrenal Insufficiency” (RAI), or “Critical Illness Related Corticosteroid Insufficiency” (CIRCI), and form part of the rationale for trials of glucocorticoid treatment in septic shock. Diagnostic criteria for these conditions have been based on plasma cortisol profiles and have proven notoriously difficult to establish. The uncertainty in this area arises from the inability of current tests to clearly identify who is truly glucocorticoid “deficient” at a cellular level, and hence who requires supplemental glucocorticoid administration. Emerging data suggest that there may be abnormalities in the tissue activity of glucocorticoids in patients with severe sepsis and plasma profiles may not be reliable indicators of tissue glucocorticoid activity, We put forward an alternative point of view, that is the spectrum of adrenocortical dysfunction in sepsis – plasma and tissue, can be grouped under the umbrella of a “sick euadrenal syndrome” rather than an adrenocortical insufficiency. [Copyright &y& Elsevier]

Background: Newborn infants have low serum cortisol concentrations due to immature Hypothalamic-Pituitary-Adrenal axis. Cortisol is highly protein bound. Hypoproteinemia, often present in sick newborns, can alter the measured total serum cortisol (TC) without affecting free cortisol (FC) concentrations. Objective: Evaluate the relationship between baseline FC and TC in sick newborn infants with refractory hypotension. Methods: Simultaneous baseline FC and TC were measured in neonates with refractory hypotension. TC was measured using radioimmunoassay and FC by LC/MS/MS. The correlation between the concentration pairs was determined using regression. Results: Thirty five of 36 pairs with patients serum cortisol concentrations were included. One pair was removed due to being a strong outlier. The mean ± SD gestational age and birth weight were 29.3 ± 5.7 weeks and 1.52 ± 1.28 kg, respectively. FC (0.02 to 42.5 mcg/dL) and TC (0.7 - 33.2 mcg/dL) varied widely. The overall correlation between FC and TC was r = 0.48 (p = 0.003). When analyzed based on TC ≤ or > 10 mcg/dL the correlation weakened (r = 0.38 and r = 0.21, respectively). For infants ≤34 and >34 weeks correlation were similar (r = 0.48 and 0.45, respectively). Conclusions: In sick newborns, correlation between FC and TC is weak. TC measured routinely may not reflect the extent of adrenal insufficiency. [ABSTRACT FROM AUTHOR]

Abstract: Background: Free cortisol (FC) can be calculated from measurements of total cortisol and binding proteins or measured after mechanical separation of unbound and bound fractions by equilibrium dialysis or ultrafiltration. FC can then be measured indirectly by 3H-cortisol dilution or directly by immunologic or tandem mass spectrometry assays. Methods: We compared FC measured with ultra high performance liquid chromatography – tandem mass spectrometry (UHPLC MS/MS) with 3H-cortisol dilution in ultrafiltrates and dialysates and also with calculated FC (Coolens equation). An adult FC reference interval was established. Results: The UHPLC MS/MS and 3H-cortisol dilution methods were non-linearly related (Cusum linearity test p <0.001) but well correlated (R 2 =0.984). FC calculated with Coolens equation agreed with the UHPLC MS/MS method. Impurity of 3H-cortisol and non-specific adsorption were excluded as causes on non-linearity. Ultrafiltration was linearly related to equilibrium dialysis, simpler to perform and more repeatable. A gender non-specific FC reference interval of 2.1–19.1nmol/L was established. Conclusions: In view of the non-linearity between measuring techniques and the variability of reported reference ranges, care should be exercised in adopting a reference range. The ultrafiltration UHPLC MS/MS method we described is robust and suitable for use in a routine laboratory. [Copyright &y& Elsevier]

Purpose: Corticosteroid insufficiency may occur among critically ill patients, but the diagnosis remains controversial. Historically assessment of free cortisol (FC) by means of equilibrium dialysis (ED) has required large blood volumes and prolonged fractionation time preceding analysis. We hypothesized that temperature-controlled centrifugal ultrafiltration with chemiluminescence immunoassay (CU/CI) would provide real-time FC data that highly correlated with ED/radioimmunoassay (ED/RI) or liquid chromatography/mass spectrometry (LC/MS) techniques. Methods: We quantified and correlated baseline and corticotropin-stimulated TC and FC by means of CU/CI, ED/RI, and LC/MS among healthy adults and 37 critically ill children. Results: Among critically ill children, FC was three- to fivefold higher than the healthy adults at baseline and increased another five- to eightfold following corticotropin administration. While TC increased approximately twofold following corticotropin administration, FC increased on average more than eightfold. Serum FC per CU/CI highly correlated with FC per ED/RI or LC/MS, but results were available in a fraction of the time. Children failing to increase TC by >9.0 μg/dL (248 nM) following corticotropin demonstrated an appropriate FC increase. Nearly 50% of critically ill children exhibited FC <2.0 μg/dL (55 nM). Neither FC nor TC concentrations correlated significantly with measures of illness severity. Conclusions: Quantification of FC utilizing CU/CI was fast (1-2 h) and results correlated highly with ED/RI or LC/MS methodologies. These data require validation with larger cohorts of healthy and critically ill children but indicate that real-time FC quantification is available to guide cortisol replacement therapy. [ABSTRACT FROM AUTHOR]

Background & Aims: Adrenal insufficiency is a common disorder among cirrhotic patients. Adrenal function is usually assessed with serum total cortisol assays. Free cortisol (active fraction) represents only 10% of serum total cortisol, the remaining 90% being linked to cortisol-binding globulin (CBG) and albumin. In cirrhotic patients, the synthesis of these proteins is reduced, which could lead to an overestimation of the prevalence of adrenal insufficiency. Salivary cortisol assessment adequately reflects free cortisol plasma concentration. However, this method has never been validated in cirrhotic patients. The objectives of this report were to assess the following parameters by a prospective observational study: (1) correlation between salivary, serum total and free cortisol, (2) adrenal insufficiency prevalence using salivary and serum assays, (3) parameters associated with a discrepancy between both tests, and (4) adrenal insufficiency risk factors among cirrhotic patients. Methods: Salivary and serum total cortisol were assessed before and 1h following an injection of corticotropin (250 μg) in patients hospitalized for cirrhosis complications without shock. CBG was measured and free cortisol was assessed by the Coolens formula. Results: Eighty-eight patients were included in the study (Child–Pugh C: 68.2%). Free cortisol was more strongly correlated with salivary than with serum total cortisol (Spearman coefficient=0.91 vs. 0.76, respectively, p <0.001). Among included patients, 9.1% had adrenal insufficiency according to salivary cortisol and 33.0% had adrenal insufficiency according to serum total cortisol (p =0.001). Hypoalbuminemia was the only factor associated with a discrepancy between the results of both tests. Adrenal insufficiency risk factors were ascites and low HDL-cholesterol plasma concentration. Conclusion: Using serum total cortisol assays overstate adrenal insufficiency prevalence among cirrhotic patients, mainly because of inaccurate concentrations related to hypoalbuminemia. Salivary cortisol assays should be preferably used in these patients. [Copyright &y& Elsevier]

Abstract: Many studies have recently focused on stress as a marker of an animal’s well being. Since animals respond to a stressor by increasing their glucocorticoid (GC) levels there has been much interest in measuring these hormones. Fecal GC analyses have been used in a wide range of studies as they are an easily obtained, non-invasive measure of these stress hormones. However, these analyses rest on two major assumptions. First, they assume that fecal GC metabolites reflect free, biologically active levels of GCs in the plasma. Second, they assume that differences in fecal GC metabolite levels among animals are an accurate reflection of their physiological state and thus of their ability to respond to a stressor. We tested these assumptions in a population of free-ranging snowshoe hares (Lepus americanus) in the southwestern Yukon, from 2006 to 2008. Both assumptions were verified. Plasma free cortisol levels mirrored bile and fecal cortisol metabolite (FCM) levels, but plasma total cortisol levels did not. Differences in FCM concentrations among hares robustly predicted their response to a hormonal challenge. Hares with higher FCM concentrations showed a greater resistance to the suppression of their free plasma cortisol following a dexamethasone injection and a more marked increase of free plasma cortisol following an ACTH injection. Furthermore, we found that changes in FCM concentrations in autumn and winter over two years reliably tracked changes in plasma free cortisol levels obtained from the hormonal challenge test. These results indicate that both fecal and plasma measures of an animal’s stress physiology are concordant: they tell the same story. [Copyright &y& Elsevier]

Melatonin, which shows a robust nycthemeral rhythm, plays the role of an endogenous synchronizer, able to stabilize and reinforce circadian rhythms and maintain their mutual phase relationships. Additionally, melatonin is a potent antioxidant and displays immunological properties. Because free radical generation, immune dysfunction, and sleep and metabolic disorders are involved in the short- and long-term pathophysiology of the burn syndrome, we undertook the study of daily urine melatonin, 6-sulfatoxymelatonin (aMT6s, the main hepatic melatonin metabolite), and cortisol variations plus temperature profiles in burn patients using a non-invasive protocol. Eight patients (6 males, 2 females) were studied on three occasions after admission to the intensive care unit (early session: days 1 to 3; intermediate session: day 10; late session: days 20 to 30). Melatonin, aMT6s, and free cortisol levels were determined in urine samples collected at 4 h intervals over a continuous 24 h span. Core temperature was recorded daily. Controls consisted of healthy subjects in the same age range. Cosinor analysis of the data provided an evaluation of mesor, amplitude, and acrophase of circadian rhythms. Also, we calculated day (D), night (N), and 24 h hormone excretions, N/D ratio for melatonin and aMT6s, and D/N ratio for cortisol. These data were analyzed using Kruskal-Wallis test followed by multiple comparisons. Cosinor analysis did not detect a circadian rhythm in melatonin, aMT6s, or cortisol in any of the three sessions. D melatonin excretion displayed a major increase, resulting in a decreased N/D melatonin ratio, and the melatonin mesor (24 h mean) was increased in the early session, compared with controls. For aMT6s, only the early N/D ratio was decreased, and the mesor of the intermediate session increased. These results were not the consequence of hepatic and/or kidney alteration, as the patients' hepatic and renal parameters were in the normal range. The D and N melatonin/aMT6s ratios of controls and patients were similar, and the aMT6s profiles were superimposed on the melatonin ones, mainly during the day. The D, N, and 24 h cortisol values were increased in all sessions, except for the D level of the early session. The consistently increased mesors in the three sessions provided confirmation. The core temperature profiles were abnormal in all three sessions, mainly during the night, although there was a tendency toward normalization with time. The individual mesors were consistently increased compared with controls. Globally, the abnormalities we report could participate in the pathophysiology of short- and long-term alterations observed in burn syndrome, especially disturbances of sleep, metabolism, and immune function. (Author correspondence: ). [ABSTRACT FROM AUTHOR]

In critical illness, serum total cortisol (TC) may not adequately reflect adrenal function because of reduced cortisol-binding globulin (CBG). To evaluate adrenal function of critically ill children, using free cortisol index (FCI), calculated free cortisol (cFC), and TC levels. Thirty-two critically ill and 36 healthy children were included. All children underwent the 1 μg cosyntropin test. TC and CBG levels were measured. Basal and peak TC, FCI, and cFC were determined. Basal and peak TC, FCI, and cFC of critically ill children were significantly higher than those of the controls. Compared with TC, both basal and peak FCI and cFC of the patients were higher than those of controls to a greater degree. Use of FCI or cFC to diagnose adrenal insufficiency (AI) reduced the frequency of diagnosis of AI by 50%. FCI and cFC better reflect the dynamic changes of adrenal function of critically ill children. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: : To develop, optimize, and validate a generalized mass action, equilibrium solution that incorporates measured concentrations of albumin as well as cortisol binding globulin (CBG) to estimate free cortisol. Design and methods: : Free cortisol was estimated by Coolens method or by cubic equilibrium equation and compared to measured free cortisol, determined by ultrafiltration method, in subjects with septic shock (n =45), sepsis (n =19), and healthy controls (n =10) at 0, 30, and 60 min following administration of cosyntropin (250 mcg). The data set also included repeat testing in 30 subjects following recovery from sepsis/septic shock. The equilibrium dissociation constant for cortisol binding to albumin (K A) was optimized by non-linear regression. The cubic equilibrium solution was also used to model the influence of cortisol, CBG, and albumin concentration on free cortisol. Results: : Compared to measured free cortisol, the cubic solution, using an optimized K A of 137,800 nM, was less biased than Coolens solution, with mean percent error of −23.0% vs. −41.1% (paired t test, P <0.001). Standard deviation values were also significantly lower (Wilks'' test, P <0.001) for the cubic solution (SD 35.8% vs. 40.8% for cubic vs. Coolens, respectively). Modeling studies using the cubic solution suggest an interaction effect by which low concentrations of CBG and albumin contribute to a greater increase in free cortisol than the sum of their independent effects. Conclusions: : Mass action solutions that incorporate the measured concentration of albumin as well as CBG provide a reasonably accurate estimate of free cortisol that generalizes to conditions of health as well as a setting of hypercortisolism and low CBG and albumin concentrations associated with septic shock. Modeling studies emphasize the significant contribution of albumin deficiency and albumin-bound cortisol under conditions of CBG-deficiency, and identify a synergistic effect by which combined CBG and albumin deficiency contribute to elevation of free cortisol in septic shock. [Copyright &y& Elsevier]

Abstract: It is generally considered that stress causes decreased immune function in fish. In this study we examined in Atlantic salmon (Salmo salar Linnaeus) the effects of both short- (a single 15s out of water) and long-term (4weeks of daily handling 15s out of water) stress on plasma cortisol (free and total) and glucose levels, expression of interleukin-1β (IL-1β) and survival of head kidney (HK) macrophages under culture with Aeromonas salmonicida. In the short-term study, samples were collected prior to the application of the stressor, and at 1, 3, 6, 12 and 24h post stress. Free and total plasma cortisol levels and the percentage of free cortisol increased significantly in the stressed group at 1 and 3h post stress. Plasma glucose levels were significantly higher than those of control fish at 1, 3 and 6h post stress. Constitutive expression of IL-1β in macrophages isolated from head kidneys in stressed fish was significantly higher at 1 and 3h post stress. However, lipopolysaccharide (LPS) stimulated expression of IL-1β in HK macrophages, exhibited significantly higher fold increases in unstressed fish compared to stressed fish. In the long-term study, with the exception of an increase in plasma glucose levels at 1week, there were no significant differences in stress parameters between groups. There was a significantly higher constitutive IL-1β expression in macrophages isolated from stressed fish over the first 2weeks. At weeks 1, 2 and 3 the magnitude of IL-1β response of isolated HK macrophages to LPS stimulation was reduced in >90% of the stressed fish. At 4weeks there was no significant difference in inducible IL-1β expression between the groups. Macrophages isolated from stressed fish also showed significantly decreased survival when exposed to A. salmonicida. This study shows a clear pattern from repeated handling stress, whereby effects on immune cells begin with increased constitutive expression of IL-1β, followed by decreased stimulation of leucocytes by extracellular antigen, and finally decreased leukocyte survival when exposed to A. salmonicida. The implications of these changes in the immune system will be discussed with respect to the use of classical indicators of stress to predict possible effects on the immune system of fish. [Copyright &y& Elsevier]

The objective of this study was to evaluate the influence of lowdose combined oral contraception (COC) on basal and stimulated (1 μg ACTH test) levels of serum and salivary cortisol (F), cortisone and on basal serum cortisol binding globulin (CBG), adrenocorticotropic hormone (ACTH), dehydroepiadrosterone (DHEA) and calculated free cortisol in healthy young women. Three-month administration of COC resulted in 1) significant increase of basal (454.0±125.0 to 860.9±179.7 nmol/l) and ACTH-stimulated serum cortisol in 30th min (652.3±60.5 to 1374.1±240.6 nmol/l); 2) no significant change of basal (15.4±7.3 to 18.9±8.5 nmol/l) and ACTH-stimulated salivary cortisol at the 30th min (32.4±8.8 to 32.9±9.0 nmol/l); 3) no significant change of basal serum cortisone (38,8±7.68 to 45.2±24.2 nmol/l) and ACTH-stimulated cortisone at the 30th (34.8±10.9 to 47.0±35.7 nmol/l); 4) significant increase of basal ACTH (17.2±9.0 to 38.2±29.4 ng/l), CBG (991.0±161.0 to 2332.0±428.0 nmol/l), and 5) no significant change of basal DHEA (24.6±15.7 to 22.6±11.7μmol/l) and calculated basal value for free cortisol (22.8±14.9 to 19.2±6.9 nmol/l). In conclusions, higher basal and ACTH-stimulated serum cortisol were found after three-month administration of COC, while basal and stimulated salivary cortisol were not significantly affected. Therefore, salivary cortisol can be used for assessment of adrenal function in women regularly using COC. [ABSTRACT FROM AUTHOR]

To date, a single report has appeared on the use of salivary cortisol for adrenal function testing with a low dose ACTH, although 1 µg has become preferred as a more physiological stimulus than the commonly used 250 µg ACTH test. Our present study was aimed to obtain physiological data on changes of free salivary cortisol after 1 µg ACTH stimulation. This approach was compared with the common method based on the changes of total serum cortisol. Intravenous, lowdose ACTH test was performed in 15 healthy women (aged 22-40 years) with normal body weight, not using hormonal contraceptives, in the follicular phase of the menstrual cycle. Blood and saliva for determination of cortisol were collected before ACTH administration and 30 and 60 min after ACTH administration. Basal concentration of salivary cortisol (mean ± S.E.M., 15.9±1.96 nmol/l) increased after 1 µg ACTH to 29.1±2.01 nmol/l after 30 min, and to 27.4±2.15 nmol/l after 60 min. The differences between basal and stimulated values were highly significant (p<0.0001). The values of salivary cortisol displayed very little interindividual variability (p<0.04) in contrast to total serum cortisol values (p<0.0001) A comparison of areas under the curve (AUC) related to initial values indicated significantly higher AUC values for salivary cortisol than for total serum cortisol (1.89±0.88 vs. 1.22±0.19, p<0.01). Correlation analysis of serum and salivary cortisol levels showed a borderline relationship between basal levels (r=0.5183, p=0.0525); correlations after stimulation were not significant. Low-dose ACTH administration appeared as a sufficient stimulus for increasing salivary cortisol to a range considered as a normal adrenal functional reserve. [ABSTRACT FROM AUTHOR]

Abstract: Background: Measurement of cortisol is of particular importance for early diagnosis of adrenocorticotropic dysfunction and therapeutic monitoring. Saliva samples clearly present an easily manageable, non-invasive and anytime practicable assessment method of glucocorticoid production. The aims of the present study were to compare salivary cortisol collection method “Salivette” versus “passive drool”, salivary cortisol concentrations with calculated free serum cortisol (FSC) and total serum cortisol (TSC) and the prediction of serum cortisol by salivary cortisol. Methods: Saliva samples were collected by Sarstedt-Salivette® and by passive drooling into plastic tubes. Salivary cortisol, TSC measurements and calculation of FSC concentrations as well were carried out simultaneously in 10 healthy volunteers on two consecutive days at six definite points of time (08.00–23.00). Results: Significant differences of salivary cortisol concentrations dependent on the saliva collection method (F-test 6.45; p =0.0317) and points of times were found (F-test 41.19; p <0.0001). Stronger overall correlation was observed in Salivette-salivary cortisol vs. TSC (r =0.813) and FSC (r =0.836) as compared to passive drooling. Specifically, more significant correlations were established in Salivettes vs. TSC (8/12 points of time, 66.7%) and FSC (8/12 points of time, 66.7%) than in passive drooling (TSC: 1/12 points of time, 8.3% and FSC: 2/12 points of time, 16.7%). Importantly, cortisol in Salivettes was a better predictor for FSC and TSC than passive drooling. Conclusions: Our data indicate that salivary cortisol measurement with Salivettes is a reliable prediction method of total and calculated free serum cortisol levels. Moreover, it is a convenient method for saliva collection, handling and laboratory processing. [Copyright &y& Elsevier]