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What is Corticosterone?
Corticosterone is a steroid hormone belonging to the class of glucocorticoids, which are essential for regulating a variety of physiological functions in many animal species. Its chemical structure is characterized by 21 carbon atoms, and it is often referred to by its IUPAC name, 17-deoxycortisol or 11β,21-dihydroprogesterone. Corticosterone plays a critical role in the body's response to stress, metabolism, immune function, and overall homeostasis.
Chemical Structure and Synthesis
Corticosterone is synthesized from pregnenolone, a precursor derived from cholesterol, through a series of enzymatic reactions that occur primarily in the adrenal cortex. This synthesis involves several key enzymes, including 21-hydroxylase and 11β-hydroxylase, which convert pregnenolone into corticosterone and subsequently into cortisol in some species. In humans, corticosterone serves mainly as an intermediate in the synthesis of cortisol and aldosterone.
Functions of Corticosterone
Stress Response: Corticosterone is often termed a stress hormone due to its elevation during stressful situations. It supports the "fight or flight" response by increasing glucose availability in the bloodstream, providing energy to essential organs and muscles.
Metabolism Regulation: It promotes gluconeogenesis, producing glucose from non-carbohydrate sources, while also influencing lipid and protein metabolism to ensure energy availability during increased demand.
Immune Function: Corticosterone modulates the immune system by regulating inflammatory responses. It suppresses pro-inflammatory cytokine production, preventing excessive tissue damage during immune reactions. However, prolonged elevation can lead to immune suppression.
Behavioral and Cognitive Effects: The hormone affects memory and learning, particularly under stress, where elevated levels can impair cognitive function and emotional memory.
Homeostasis: Corticosterone contributes to maintaining homeostasis by regulating blood pressure and electrolyte balance, although its mineralocorticoid activity is weaker than that of aldosterone.
Corticosterone Analysis Offered by Creative Proteomics
Creative Proteomics provides comprehensive corticosterone analysis services tailored for researchers and institutions seeking to understand the biological implications of corticosterone levels.
Quantitative Measurement of Corticosterone Levels: Accurate quantification of corticosterone in biological samples such as serum, plasma, and tissue extracts to support metabolic and physiological studies.
Corticosterone Dynamics in Animal Models: Analysis of corticosterone fluctuations in response to various experimental conditions, aiding in the understanding of stress responses and endocrine regulation in different species.
Assessment of Stress-Induced Corticosterone Changes: Evaluation of corticosterone levels following exposure to stressors, facilitating research into the physiological effects of stress on animal behavior and health.
Longitudinal Studies of Corticosterone Levels: Monitoring corticosterone over time in specific cohorts to examine the long-term effects of environmental changes, stressors, or experimental treatments.
Custom Protocol Development for Corticosterone Measurement: Tailored standard operating procedures (SOPs) designed to meet specific research needs, ensuring that all analysis is aligned with project requirements.
List of Corticosterone We Can Detect
| Corticosterone Form | Description | Sample Types |
|---|---|---|
| Corticosterone (C21H30O4) | The primary form, involved in stress response and metabolism. | Serum, Plasma, Tissue Extracts |
| Corticosterone Glucuronide | A conjugated form of corticosterone, reflecting metabolism and excretion. | Urine, Serum |
| Corticosterone Sulfate | A sulfate conjugate, indicating hormonal activity and clearance. | Urine, Serum |
| Corticosterone Metabolites | Various metabolites formed during the metabolism of corticosterone. | Tissue Extracts, Urine |
Brochures
Metabolomics Services
We provide unbiased non-targeted metabolomics and precise targeted metabolomics services to unravel the secrets of biological processes.
Our untargeted approach identifies and screens for differential metabolites, which are confirmed by standard methods. Follow-up targeted metabolomics studies validate important findings and support biomarker development.
Download our brochure to learn more about our solutions.
Technology Platforms for Corticosterone Analysis
High-Performance Liquid Chromatography (HPLC)
- Instrument: Agilent 1260 Infinity HPLC System
- Detection Method: UV detection at 240 nm
- Advantages: This method offers high sensitivity, specificity, and reproducibility for corticosterone quantification in complex biological samples.
Liquid Chromatography-Mass Spectrometry (LC-MS/MS)
- Instrument: AB Sciex Triple Quad 6500+
- Detection Method: Multiple Reaction Monitoring (MRM)
- Advantages: This platform allows for the simultaneous detection and quantification of corticosterone and its metabolites with exceptional precision.
Sample Requirements for Corticosterone Analysis
| Sample Type | Untargeted Metabolomics | Targeted Metabolomics | Lipidomics | Metabolic Flux |
|---|---|---|---|---|
| Animal Tissue | 100-200 mg | 100-200 mg | 100-200 mg | |
| Plant Tissue | 100-200 mg | 100-200 mg | 100-200 mg | |
| Plasma/Serum | >100 μL | >100 μL | >100 μL | |
| Urine | 200-500 μL | 200-500 μL | 200-500 μL | |
| Saliva, Amniotic fluid, Bile, Tears, etc. | >200 μL | >200 μL | >200 μL | |
| Cells | >1*107 | >1*107 | >1*107 | >1*107 |
| Culture Supernatant | >2 mL | >2 mL | >2 mL | |
| Wastewater/Culture Medium | >2 mL | >2 mL | >2 mL | |
| Microbial Culture | >2 mL | >2 mL | >2 mL | |
| Feces/Intestinal Contents | 100-200 mg | 100-200 mg | 100-200 mg | |
| Soil Sample | >1 g | >1 g | >1 g | |
| Swab | 2 | 2 |
PCA chart
PLS-DA point cloud diagram
Plot of multiplicative change volcanoes
Metabolite variation box plot
Pearson correlation heat map
Determination of adrenaline, noradrenaline and corticosterone in rodent blood by ion pair reversed phase UHPLC–MS/MS.
Journal: Journal of Chromatography B
Published: 2018
Background
Noradrenaline, adrenaline, and corticosterone are crucial hormones involved in the stress response in rodents. The analysis of catecholamines (adrenaline and noradrenaline) is challenging due to their low concentrations and chemical instability, necessitating sensitive and selective bioanalytical methods. In contrast, corticosterone is more stable and found at moderate levels in rodent blood. Techniques such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) and ultra high-performance liquid chromatography (UHPLC) have gained popularity for their enhanced sensitivity and resolution. This study aims to develop a robust UHPLC-MS/MS method to simultaneously measure noradrenaline, adrenaline, and corticosterone using ion pair chromatography, demonstrating improved peak response by incorporating ion pair reagents in both the mobile phase and sample reconstitution solvent.
Materials & Methods
Chemicals and Materials
Adrenaline, corticosterone, and their isotopically labeled standards were obtained from Sigma-Aldrich and C/D/N Isotopes. Reagents included formic acid and acetonitrile, sourced from VWR and Lab-Scan, respectively.
Preparation of Calibrators and QC Samples
Stock solutions of corticosterone and internal standards were made in pure methanol. Calibration and quality control samples ranged from 1.25 to 2000 nM and 1.00 to 1600 nM, respectively, using 25 mM formic acid.
Rodent Blood Sample Collection
Blood samples were collected from male Sprague Dawley rats using heparin to prevent clotting. Samples for corticosterone analysis were processed via UHPLC-MS/MS within two hours or stored at -80°C for stability testing.
Sample Preparation for Corticosterone Analysis
Rodent blood (300 µL) was mixed with 25 mM formic acid and internal standards, followed by liquid-liquid extraction with acetonitrile/methanol/formic acid. Samples were dried and reconstituted in 40 mM HFBA in 25 mM FA for UHPLC-MS/MS analysis.
Metabolomics Analysis Instrumentation
Analysis was performed using an Acquity™ UPLC system with a Xevo-TQS mass spectrometer. Chromatography utilized a gradient suitable for separating corticosterone.
Method Validation
The method was validated for calibration curves, precision, and recovery of corticosterone in rodent and human blood.
Stability Determination for Corticosterone in Metabolomics
Stability was assessed in pooled rat blood samples with recovery defined as within 100 ± 20% after storage at -80°C.
Results
The study successfully established the Lower Limit of Quantification (LLOQ) and Limit of Detection (LOD) for the analytes. The LLOQ was determined at a QC concentration where intermediate precision was ≤ 20% and bias was within ± 20%, requiring a signal-to-noise ratio (S/N) of ≥ 10. The LOD was defined as the QC concentration with S/N > 3. The LOD values were 1.0 nM for corticosterone and ≤ 1.0 nM for noradrenaline and adrenaline, while the LLOQ values were 1.0 nM for noradrenaline and adrenaline and 2.0 nM for corticosterone.
Matrix effects (ME) were evaluated at three concentration levels, showing that adrenaline and noradrenaline were significantly affected, with ME values of 40-42% and 8%, respectively, while corticosterone showed less impact at 56-71%. Corrected ME values with internal standards (ISs) were within 95-104% for adrenaline and corticosterone and 117-123% for noradrenaline, indicating that ISs effectively compensated for matrix effects.
Total ion chromatogram of noradrenaline, adrenaline, and corticosterone in standard samples prepared in 25 mM FA or in 40 mM HFBA in 25 mM FA.
Recovery rates for the analytes were measured pre- and post-sample preparation, revealing a recovery of 64-65% for corticosterone and only 12% for adrenaline and 13-15% for noradrenaline, indicating major losses during protein precipitation. Carry-over assessments indicated that carry-over was < 0.1% for all analytes, suggesting that this was not a concern.
MRM chromatograms of 0.17 µM noradrenaline and noradrenaline-d6 in spiked human blood samples reconstituted in either 25 mM FA (a) or 25 mM FA containing 10-80 mM HFBA concentrations (b-d).
Stability studies using isotopic labeled surrogate analytes demonstrated that they remained stable under various conditions, including 45 minutes on ice, two freeze/thaw cycles, and one week at -80°C. The method was further validated for robustness by showing that the addition of HFBA to the mobile phase did not adversely affect instrument performance over 200 injections.
The application of the developed method revealed the basal levels of adrenaline, noradrenaline, and corticosterone in rat blood to be 13 ± 0.6 nM, 15 ± 0.7 nM, and 9.4 ± 0.4 nM, respectively. In mouse blood samples, significant increases in corticosterone were observed after stress exposure, while no significant changes in noradrenaline levels were detected, and adrenaline levels could not be reported due to low S/N ratios. Overall, the method proved reliable for quantifying catecholamines and corticosterone in blood samples while addressing critical analytical parameters.
Reference
- Bergh, Marianne Skov-Skov, et al. "Determination of adrenaline, noradrenaline and corticosterone in rodent blood by ion pair reversed phase UHPLC–MS/MS." Journal of Chromatography B 1072 (2018): 161-172.
How can I determine the appropriate time point for sample collection in my study?
The timing of sample collection for corticosterone analysis is critical, especially in studies involving stress responses or circadian rhythms. Ideally, samples should be collected at consistent times relative to stressor exposure or based on the organism's natural activity cycle. For example, in diurnal species, samples taken in the early morning may reflect baseline levels, while samples collected shortly after a stressor can capture peak responses. We recommend conducting preliminary studies to establish the best time points for your specific research context.
What are the potential sources of variability in corticosterone measurements?
Several factors can influence corticosterone levels, including individual differences (age, sex, health status), environmental conditions (temperature, housing), and the specific stressors used in experiments. Additionally, variations in sample handling and processing (e.g., freezing times, extraction methods) can affect results. To minimize variability, it's essential to standardize these conditions as much as possible and include appropriate controls in your experimental design.
Can corticosterone analysis be performed in combination with other hormonal assays?
Yes, combining corticosterone analysis with other hormonal assays, such as cortisol or aldosterone, can provide a more comprehensive understanding of the endocrine response to stress. When planning such studies, it is crucial to consider potential interactions between hormones and the assay techniques used. We can provide guidance on optimizing sample collection and analysis protocols to facilitate multi-hormone studies.
What statistical methods are recommended for analyzing corticosterone data?
The analysis of corticosterone data often requires statistical approaches that account for repeated measures or multiple comparisons, especially in longitudinal studies. Common methods include mixed-effects models or repeated measures ANOVA, which can accommodate within-subject variability. Additionally, it's essential to check assumptions of normality and homogeneity of variance. Consulting with a biostatistician during the study design phase can help in selecting the most appropriate statistical methods.
How does the choice of sample matrix affect corticosterone analysis?
Different biological matrices can yield varying corticosterone levels and insights into hormonal dynamics. For instance, serum samples may reflect immediate endocrine responses, while urine samples provide a cumulative measure of corticosterone excretion. It's important to consider what physiological context is most relevant to your research question. We recommend discussing your study's objectives with our team to determine the best sample type for your specific needs.
What extraction methods do you use for corticosterone from biological samples?
We typically employ solid-phase extraction (SPE) and liquid-liquid extraction (LLE) methods for isolating corticosterone from biological samples. SPE is favored for its efficiency and ability to concentrate the analyte while removing potential interferences. The choice of method can depend on the sample type and the specific goals of your analysis. We can work with you to select the most suitable extraction technique based on your experimental design.
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