Different Concentrations Of Cortisol Research Articles

Fast and reliable quantification of aldosterone, cortisol and cortisone via LC-MS/MS to study 11β-hydroxysteroid dehydrogenase activities in primary cell cultures

Cell culture experiments can support characterization of enzymatic activities in healthy and tumorous human tissues. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) enables simultaneous measurement of several steroids from a single sample, facilitating analysis of molecular pathways involved in steroid biosynthesis. We developed a reliable but fast method for quantification of cortisol, cortisone and aldosterone in cell culture supernatant. Validation, including investigation of matrix-matched calibration, was performed for two different cell types. Utility of the method was demonstrated in the study of 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) activity under conditions of glucocorticoid and mineralocorticoid excess in different cell types. Aldosterone, cortisol and cortisone were extracted by liquid-liquid extraction (LLE) with methyl tert-butyl ether from 1mL of cell culture supernatant. Steroids were separated on a Kinetex biphenyl column (50 ×2.1mm, 2.6µm) with gradient elution of water and methanol containing 2mM ammonium format and analysed in multiple reaction monitoring mode after positive electrospray ionization. Application of the method included cell culture experiments with two different primary cell types, human coronary artery smooth muscle cells (HCSMC) and human coronary artery endothelial cells (EC). Cells were treated with different concentrations of cortisol, aldosterone and mifepristone, a glucocorticoid receptor antagonist and quantitative PCR was performed. The method exhibits high precision (CV ≤ 6%) and accuracy (deviation from nominal concentration ≤ 6%) for concentrations above the limit of quantification (LoQ) which is 0.11, 0.56 and 0.69 nmol/L for aldosterone, cortisone and cortisol, respectively. Calibration curves did not differ when prepared in media or solvent. The method enabled us to confirm activity of HSD11B2 and concentration dependent conversion of cortisol to cortisone in HCSMC (median conversion ratio at 140nM cortisol = 1.46%). In contrast we did not observe any HSD11B2 activity in EC. Neither addition of high aldosterone, nor addition of 1µM mifepristone had impact on glucocorticoid concentrations. Quantitative PCR revealed expression of HSD11B1 and HSD11B2 in HCSMC but not in EC. We present a fast and reliable method for quantification of cortisol, cortisone and aldosterone in cell culture supernatants. The method enabled us to study HSD11B2 activity in two different cell types and will support future experiments investigating mechanisms of target organ damage in conditions of glucocorticoid and mineralocorticoid excess.

Machine learning guided electrochemical sensor for passive sweat cortisol detection

A noninvasive sensor was developed on a flexible, nano-porous substrate with the capability to detect cortisol passively from human sweat. A Machine Learning (ML) algorithm was also developed to analyze sensor data to detect rising and falling trends of cortisol with time, which can potentially inform the wearer of rising or falling cortisol levels. Sensor response was measured by Electrochemical Impedance Spectroscopy (EIS) assay with spiked synthetic sweat within the physiological concentration range of cortisol, for which responses to low, medium, and high concentrations were significant. Calibration studies established a dynamic range of 8–140 ng/ml and EIS demonstrated a dose-dependent response and operational frequency range of 100–500 Hz. Inter-assay and intra-assay variations were below 20%. One-way ANOVA and post hoc Tukey test established significant response (p < 0.05) to different concentrations of cortisol. EIS was performed with dosing ranging from high-low and low-high concentrations of cortisol, to simulate rising and falling cortisol levels over time. Rate of change of response to shifts in cortisol concentration was used to train a weighted K nearest neighbor (KNN) machine learning algorithm to detect and classify increasing and decreasing cortisol in sweat. Accuracy was validated to be 100% by k means cross validation. Sensor analysis of sweat samples from human subjects showed a 100% accuracy in testing with the algorithm along with a 100% True positive and 0% False negative rate, thus showing that a wearable sweat sensor can be used with ML to detect rising and falling trends in cortisol for on demand, noninvasive sensing.

On The Application of SiO2/SiC Grating on Ag for High-Performance Fiber Optic Plasmonic Sensing of Cortisol Concentration.

This paper reports on high-accuracy simulation of a grating structure based fiber optic plasmonic sensor for salivary cortisol sensing. Gratings of SiO2 and SiC (one at a time) in combination with a thin Ag layer are considered to be in direct contact with analyte medium (solutions containing different concentrations of cortisol) considering that the groove regions are also filled with analyte. The optimization of Ag layer thickness is carried out to achieve maximum power loss (PL) corresponding to cortisol concentration variation. The variation of PL (in dB) spectra with the angle of incidence (α) is the sensing mechanism of the proposed scheme. Sensing performance is extensively analyzed in terms of sensitivity, limit-of-detection (LOD) and figure-of-merit (FOM) that incorporates both the sensitivity and the width of the corresponding PL curves. While the sensitivity and FOM values are significantly large, the results also reveal that in angular interrogation mode (AIM), an average LOD of 9.9 pg/mL and 9.8 pg/mL is obtained for SiO2 and SiC-based sensor designs, respectively. When the intensity interrogation method (IIM) in place of AIM is considered, an average LOD of 22.6 fg/mL and 68.17 fg/mL is obtained for SiO2 and SiC-based sensor designs, respectively. LOD (with IIM, in particular) is considerably better than the present-state-of-art related to cortisol monitoring. Pragmatic model for possible practical implementation of sensor scheme is also discussed. The involvement of optical fiber in the proposed sensor design makes it possible to implement it as a flexible sensor or for wearable solution for cortisol detection via sweat monitoring as well as for measuring cortisol level in aquaculture tanks where concentration levels are much lower than 10 ng/mL.

The proliferative effect of cortisol on bovine endometrial epithelial cells

BackgroundBovine endometrial epithelial cells (BEECs) undergo regular regeneration after calving. Elevated cortisol concentrations have been reported in postpartum cattle due to various stresses. However, the effects of the physiological level of cortisol on proliferation in BEECs have not been reported. The aim of this study was to investigate whether cortisol can influence the proliferation properties of BEECs and to clarify the possible underlying mechanism.MethodsBEECs were treated with different concentrations of cortisol (5, 15 and 30 ng/mL). The mRNA expression of various growth factors was detected by quantitative reverse transcription-polymerase chain reaction (qPCR), progression of the cell cycle in BEECs was measured using flow cytometric analysis, and the activation of the Wnt/β-catenin and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathways was detected with Western blot and immunofluorescence.ResultsCortisol treatment resulted in upregulated mRNA levels of vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF); however, it had no influence on transforming growth factor-beta1 (TGF-β1). Cortisol (15 ng/mL) accelerated the cell cycle transition from the G0/G1 to the S phase. Cortisol upregulated the expression of β-catenin, c-Myc, and cyclinD1 and promoted the phosphorylation of PI3K and AKT.ConclusionsThese results demonstrated that cortisol may promote proliferation in BEECs by increasing the expression of some growth factors and activating the Wnt/β-catenin and PI3K/AKT signaling pathways.

Microglia Activated by Excess Cortisol Induce HMGB1 Acetylation and Neuroinflammation in the Hippocampal DG Region of Mice Following Cold Exposure.

Cold stress can induce neuroinflammation in the hippocampal dentate gyrus (DG), but the mechanism underlying neuronal apoptosis induced by cold stress is not well-understood. To address this issue, male and female C57BL/6 mice were exposed to a temperature of 4 °C for 3 h per day for 1 week, and glial cell activation, neuronal apoptosis, and neuroinflammation were evaluated by western blotting, immunofluorescence, terminal deoxynucleotidyl transferase 2’-deoxyuridine 5’-triphosphate (dUTP) nick end labeling, Nissl staining, and immunohistochemistry. Additionally, BV2 cells were treated with different concentrations of cortisol (CORT) for 3 h to mimic stress and molecular changes were assessed by western blotting, immunofluorescence, and co-immunoprecipitation. We found that excess CORT activated glial cells and increased neuroinflammation in the DG of mice exposed to cold temperatures, which was associated with increased acetylation and nuclear factor-κB signaling. These effects were mediated by the acetylation of lysine 9 of histone 3 and lysine 310 of p65, which resulted in increased mitogen-activated protein kinase phosphorylation, nuclear translocation of p65, microglia activation, and acetylation of high-mobility group box 1. Neuroinflammation was more severe in male compared to female mice. These findings provide new insight into the mechanisms of the cold stress response, which can inform the development of new strategies to combat the effects of hypothermia.

Cortisol Excess-Mediated Mitochondrial Damage Induced Hippocampal Neuronal Apoptosis in Mice Following Cold Exposure.

Cold stress can induce neuronal apoptosis in the hippocampus, but the internal mechanism involving neuronal loss induced by cold stress is not clear. In vivo, male and female C57BL/6 mice were exposed to 4 °C, 3 h per day for 1 week. In vitro, HT22 cells were treated with different concentrations of cortisol (CORT) for 3 h. In vivo, CORT levels in the hippocampus were measured using ELISA, western blotting, and immunohistochemistry to assess the neuronal population and oxidation of the hippocampus. In vitro, western blotting, immunofluorescence, flow cytometry, transmission electron microscopy, and other methods were used to characterize the mechanism of mitochondrial damage induced by CORT. The phenomena of excessive CORT-mediated oxidation stress and neuronal apoptosis were shown in mouse hippocampus tissue following cold exposure, involving mitochondrial oxidative stress and endogenous apoptotic pathway activation. These processes were mediated by acetylation of lysine 9 of histone 3, resulting in upregulation involving Adenosine 5‘-monophosphate (AMP)-activated protein kinase (APMK) phosphorylation and translocation of Nrf2 to the nucleus. In addition, oxidation in male mice was more severe. These findings provide a new understanding of the underlying mechanisms of the cold stress response and explain the apoptosis process induced by CORT, which may influence the selection of animal models in future stress-related studies.

Human Serum Albumin Structure in Presence of Different Concentrations of Cortisol and Glucose: An In Vitro Modeling Under Normal and Hyperglycemic Conditions

Background: Glucose is an essential element in the supply of body’s energy. In diseases such as diabetes, glucose increase is associated with disturbance in metabolism. Cortisol is an important hormone in the regulation of glucose metabolism, and human serum albumin (HSA) is one of the most important glucose and cortisol transmitters in blood. Interaction between albumin and these ligands could affect HSA secondary structure and its stability. Objectives: The aim of this study was to investigate HSA secondary structure in the presence of different concentrations of glucose and cortisol. Methods: This was an in vitro (analytical/descriptive) study in which, completely randomized design was used to study the interaction between human serum albumin at 37°C at different glucose concentrations of 0, 80, 180, 240, and 400 mg/dL and cortisol at concentrations of 0, 10, 20, and 40 µg/dL. Intrinsic fluorescence spectroscopy and Circular dichroism (CD) were performed to obtain data. Results: HSA secondary structure underwent changes in the presence of different concentrations of cortisol and glucose. P values less than 0.01 were considered to be statistically significant. Fluorescence spectroscopy and CD results showed that at normal glucose concentrations, HSA was very flexible, beta-sheet content reduced, and the maximum increase in fluorescence and blue-shift happened. At higher concentrations of glucose, HSA became rigid. Also, HSA in presence of 10 µg/dL cortisol was very flexible; but a cortisol concentration of 40 µg/dL caused stability in HSA structure in presence of different glucose concentrations. Conclusions: Under normal glucose conditions, very low cortisol concentrations create large changes in HSA secondary structure. At normal glucose concentrations, some of the binding sites of HSA that are all occupied by glucose at higher concentrations become available to cortisol. Cortisol structure is very hydrophobic, which causes large changes in HSA secondary structure and significant increases in quenching and blue shift. In conclusion, binding of compounds such as medicines to HSA sites may be affected by competitive bindings of glucose, depending on its concentration in the blood.

Effects of Chronic Cortisol Administration on Global Expression of GR and the Liver Transcriptome in Sparus aurata

The present work was designed to assess the effects of artificially increased high plasma cortisol levels induced by slow-release cortisol implants on the mRNA abundance of the glucocorticoid receptor (GR) in different organs of Sparus aurata (Gilthead sea bream), as well as to evaluate global transcriptional changes in the liver, using the Aquagenomics S. aurata oligo-nucleotide microarray technology. For that purpose, groups of fish were intraperitoneally injected with implants containing two different concentrations of cortisol (50 or 200 μg/g body weight). Blood and organs were sampled after 7 and 14 days of cortisol implantation. Only fish with 200 μg/g implants exhibited a significant rise in plasma cortisol. Thus, we evaluated the expression of the GR in different organs in these fish 7 and 14 days post-implantation. GR mRNA abundance was upregulated in head kidney and heart of fish at both sampling times. In liver and muscle, GR mRNA abundance was upregulated after 14 days, whereas in gills, the GR mRNA transcript was upregulated earlier, at day 7. These results suggest that increased plasma cortisol induced by a slow-release implant of cortisol mimics the overall effects of stress and affects the expression of GR mRNA in a time- and organ-specific manner. Data obtained with the Aquagenomics S. aurata oligo-nucleotide microarray allowed the identification of a total of 491 cortisol-responsive transcripts and highlight the strong intensity of transcriptional modulation in liver of fish implanted with cortisol after 7 days, in contrast to that observed at day 14. Transcriptional remodeling highlighted a significant activity in carbohydrate metabolism mainly in the gluconeogenic pathway linked to downregulation of inflammatory and immune response processes in implanted fish.

Interleukin-1 β-mediated glucose uptake by chondrocytes. Inhibition by cortisol

The aim of this study was to investigate the in vitro effects of interleukin-1 beta (IL-1 beta) on cultured human articular chondrocytes from patients with osteoarthritis, by the evaluation of glucose uptake. We also investigated the inhibitory effect of cortisol on IL-1 beta-mediated glucose uptake. Experiments were performed by using 2-deoxy-D-[1-3H]glucose (2-DOG) and confluent monolayer cells at first passage. Confluent cells were also treated for 24 h with different concentrations of cortisol (10(-5), 10(-6) and 10(-7) mol/l). IL-1 beta (100 pg/ml) was added 6 h before glucose uptake studies. Glucose uptake stimulation was observed 3 h after the addition of 100 pg/ml IL-1 beta (+70%) and increased up to 24 h (+145%). The sensitivity and responsiveness of chondrocytes to IL-1 beta, studied after a 6 h association time, appeared to be dose-dependent from 0.1 pg/ml IL-1 beta (+50%) to 100 pg/ml (+130%) over basal values. The effect of the cytokine was protein synthesis-dependent, as demonstrated by using cycloheximide. Cortisol inhibited the action of IL-1 beta on glucose uptake because it reduced stimulating effects by 28% at concentrations as weak as 10(-6) mol/l. Results appeared similar when IL-1 beta and cortisol were added simultaneously 6 h before 2-DOG uptake. The rapid effect of cortisol was protein-synthesis dependent, as indicated by inhibition by cycloheximide. These results suggest that IL-1 beta stimulates chondrocyte metabolic activity. The inhibition of IL-1 beta-mediated glucose uptake is suggested for studying the anti-IL-1 effect of other anti-rheumatic drugs.

The effect of cortisol on the secretion of ACTH by anterior pituitary cells of the horse in culture.

Perifused equine anterior pituitary cells were used to investigate the effect of cortisol on the ACTH response to pulses of corticotrophin-releasing hormone (CRH; 0.01 nmol/l) and arginine vasopressin (AVP; 100 nmol/l), given for 5 min every 30 min for 690 min and ACTH measured in 5-min fractions. At the fourth pulse of secretagogue (0 min), a constant perifusion with cortisol began (0 nmol/l (control), 100, 200, 500, 5000 and 50,000 nmol/l) and continued until the ninth pulse (150 min). For each pulse of secretagogue, the amount of ACTH (pmol) secreted in response to each pulse (ACTH response area), the highest concentration of ACTH (microgram/l) measured after each pulse (peak height) and the mean ACTH in the three prepulse fractions (ACTH baseline) were determined. Data from control columns in each experiment were fitted by least squares to an exponential function to produce a mean control value for each end-point; results in all columns were expressed as a percentage of the mean control values. The addition of cortisol had a highly significant negative effect on ACTH response area, peak height and baseline at all times from +30 to +240 min (columns given cortisol compared with the mean of control column values by t-test). Analysis of variance of the data showed that the higher the cortisol concentration, the quicker the ACTH response area (P = 0.0072) and peak height (P = 0.002) decreased to < 50% of mean control, and the greater the maximum percentage change (suppression) in ACTH response area (P < 0.0001) and peak height (P < 0.0001). The maximum percentage change (suppression) in baseline was independent of cortisol concentration. At +30 min after the start of cortisol perifusion, the ACTH response area in CRH columns was significantly lower than in AVP columns (P = 0.0088), and remained lower 90 min after the end of perifusion (P = 0.0084) but the maximum percentage change (suppression) was not different between secretagogues. ACTH peak height was significantly (P < 0.0268) lower in CRH than in AVP columns (from +30 min until 180 min after the end of cortisol perifusion) and the maximum percentage change (suppression) was also greater (P = 0.0011) in CRH columns. This study shows the effect of different concentrations of cortisol on CRH- and AVP-induced ACTH secretion by equine anterior pituitary cells, and the time-course for ACTH responses to be inhibited by, and recover from, cortisol perifusion.(ABSTRACT TRUNCATED AT 400 WORDS)

Cortisol effect on (Na+ + K+)-stimulated ATPase activity and on bilayer fluidity of dog brain synaptosomal plasma membranes.

The binding of [14C]cortisol into dog brain synaptosomal plasma membranes (SPM) follows an exponential path described by the general formula y = a.ebx. The specific activity of the SPM-bound (Na+ + K+)-stimulated ATPase was linearly increased at different concentrations of cortisol. Changes in the allosteric properties of (Na+ + K+)-stimulated ATPase by fluoride (F-) (i.e. changes of Hill coefficients) indicate that cortisol increases the membrane fluidity. The fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene-labeled SPM decreased in cortisol treated SPM compared to untreated (control) SPM, which is consistent with a general increase in membrane fluidity. This increase of fluidity by cortisol may play a role in the physiological effects of this hormone in the brain.

Assessment of a solid phase ria cortisol kit

The CORTCK-125 kit was evaluated and compared with the fluorimetric technique currently used in the laboratory. The radioimmunoassay is performed in antibody coated tubes using I125-labelled Cortisol as a tracer. There was good correlation between the assays (r = 0.97) on analysing patient sera but the radioimmunoassay results were higher than expected from a specific Cortisol assay. The major interfering substances are prednisone, its metabolite prednisolone and progesterone (cross reactivities of 3%, 53% and 1.4% respectively). The reproducibility of the assay was examined at three different concentrations of Cortisol on 20–24 specimens. The intra-assay CV were 20%, 11% and 13% for concentrations of 110, 370 and 920 nmol/1 respectively. Inter-assay variation over 9 assays was 17%, 17% and 19% respectively. In comparison, the within and between CV of the fluorimetric method varied between 2–7% and 6–8% respectively. The kit is convenient and simple to use; however, the precision achieved was disappointing.

Effect of allergic processes of immediate and delayed types on the intensity of cortisol metabolism in the guinea pig liver

The intensity of cortisol metabolism was studied during perfusion of the liver in situ with solutions containing different concentrations of cortisol. Under these circumstances metabolism was shown to take place chiefly in the direction of cortisone formation. With an increase in the cortisol concentration in the perfusion fluid the intensity of its metabolism in the liver tissue also increased. During anaphylactic changes in the liver tissue the intensity of conversion of cortisol into cortisone was reduced, indicating a disturbance of the oxidation of cortisol. In experimental allergic encephalomyelitis the intensity of cortisol metabolism also was reduced.

Intracellular binding of [ 3H]cortisol and its effect on RNA polymerase activity in hypothalamus of the pig

Intracellular binding of [ 3H]cortisol in the hypothalamus of pigs and the effects of this binding on RNA polymerase activity were studied. Molecular-sieve chromatography of both the cytosol and the nuclear extract on Sephadex G-100 resulted in a peak of radioactivity eluted with the protein fraction. The very low elution volume of the nuclear steroid-protein complex indicated that the macromolecular receptor of the nuclear extract is larger than that of the cytoplasm. Results from the utilization of two different concentrations of cortisol rerevealed that the binding sites of the cytoplasm and nuclear extract became saturated at a cortisol concentration between 10 −7 M and 10 −4 M. Most of the radioactivity bound in the cytosol and nuclear extract possesed the solubility of cortisol. The nuclear extract possesed a greater percentage of bound radioactivity as cortisol than the cytoplasm. Chromatography of proteins of known molecular weights allowed approximation of molecular weights of the steroid-protein complexes. Most of the [ 3H]cortisol associated with protein in the hypothalamus cytosol was bound to protein with an elution volume which corresponded to a molecular weight of approximately 100,000. The peak of radioactivity in the nuclear extract was associated with protein which was eluted in a volume corresponding to a molecular weight slightly less than 150,000. The influence of the cytosol steroid-protein complexes on RNA polymerase activity in the nucleus was studied. RNA polymerase was significantly increased ( P < 0.05) when nuclei were incubated in the presence of cytosol steroid-protein complexes and compared to nuclei incubated with buffer alone, free cortisol or cortisol in a in albumin.