Abstract = 200 words, summary of the report, concise with background, techniques and results, conclusion.

Abstract = 200 words, summary of the report, concise with background, techniques and results, conclusion.

Introduction

Chronic stress is one of the most prevalent chronic conditions experienced globally (Manjusha et al., 2023). Chronic stress is defined as a sustained state of stress resulting from continual factors experienced in daily activities, such as work pressure, financial difficulties, or chronic illnesses (Roberts & Karatsoreos, 2021). Compared to acute stress, which is a short-term response, chronic stress causes the body's stress response to remain active for an extended period. This chronic stress response involves interactions including the nervous, endocrine, and immune systems, activating the body's 'fight or flight' response. This activation occurs through the hypothalamic-pituitary-adrenal (HPA) axis, which is responsible for the prolonged release of cortisol and other stress hormones (Knezevic et al., 2023). Chronic stress has significant implications for health, which can lead to the following issues: cardiovascular complications like myocardial infarction, weakened immunity due to increased cortisol levels, gastrointestinal issues such as exacerbation of Crohns disease, and mental health conditions like anxiety and depression (Franklin et al., 2021). Chronic stress can also impair cognitive functions, affecting memory, concentration, and decision-making abilities (Kim & Kim, 2023).

As noted earlier, cortisol is a significant glucocorticoid hormone produced in the body. When the body experiences high stress levels for an extended period, the hypothalamus releases corticotropin-releasing hormone (CRH), which causes the anterior pituitary gland to produce adrenocorticotropic hormone (ACTH). ACTH then stimulates the adrenal cortex to produce and release cortisol (Knezevic et al., 2023). Chronic stress ultimately impacts physiological processes by prolonged cortisol release, leading to extreme cortisol production and desensitisation of the HPA axis. Hair cortisol analysis is one of the approaches that measures long-term cortisol levels to assess chronic stress within the biomedical research (Russell et al., 2012). This study involved extracting and analysing hair samples using the Cortisol ELISA protocol. This method quantifies cortisol levels through antibody-based and color-based detection, ensuring precise measurement of cortisol concentrations (pg/mg) from extracted hair samples (Meyer et al., 2014).

The prevalence of conducting ELISA hair cortisol tests is critical for assessing chronic stress levels, these tests are a non-invasive method that test long-term cortisol measurement. This is particularly relevant given the rise in daily stress-related conditions, which can ultimately contribute to anxiety, depression, cardiovascular diseases, and deteriorate conditions like Addison's syndrome. For example, monitoring cortisol levels aids in early diagnosis and management of conditions like Cushing's syndrome, Addison's syndrome, chronic stress, and adrenal insufficiency by identifying abnormal cortisol patterns. One research study that utilised the ELISA hair cortisol technique analyzed data from 17 research journals and concluded a mean range of hair cortisol levels from 6.32 to 130.30 pg/mg (Igboanugo et al., 2023).

Following these findings from other studies, the aim of this study was to assess cortisol levels in 22 hair samples (RS01), 18 hair samples (RS02) using ELISA analysis, along with conducting an individual analysis. The samples were read using a plate reader set at a 655 nm filter, and the data was analysed using a standard curve with Prism GraphPad and a one-way t-test for all samples collectively and the individual sample. The significance of this experiment was to determine the relevance of ELISA analysis in relation to other studies and to compare the results based on the weight of the extracted hair cortisol.

Materials and Methods

Experiment 1 - Sample Preparation and Extraction: The hair sample preparation for analysis included securing 25-40 strands, followed by sterilising them with isopropanol wipes. Each sample was then cut to a length of 3 cm from the scalp end, weighed using a precision scale, and transferred into microcentrifuge tubes containing chrome steel beads. After homogenization in a bead mill to achieve a powdered form, HPLC grade methanol was added to the samples. The tubes were then rotated at room temperature for 24 hours to ensure thorough extraction (Ong & Kauter, 2024).

Experiment 2 - Determination of Hair cortisol using ELISA: Using the samples prepared in experiment 1, standard, positive, and negative controls, primary antibody, secondary antibody, enzyme substrate, phosphate buffer solution (PBS), and wash buffer were utilised. The controls and a serial dilution (1:1 to 1:64) were prepared with PBS and transferred in triplicate into a 96-well plate. Between adding the primary and secondary antibodies, as well as the enzyme substrate, the wells underwent multiple washes with wash buffer. Finally, the results were read using a plate reader set to a 655 nm filter (Ong & Kauter, 2024).

Data analysis: The data was analysed using a standard curve constructed with Prism GraphPad. Statistical analysis included conducting p-tests to assess significance for both the collective samples (RS01 n=22), (RS02 n=18) and an individual sample. The output of the microplate reader had to be converted to amount of cortisol per unit weight of powdered hair, this conversion utilised the following formula was used: (A/B) x (C/D) x E x 10,000 (pg/mg).

Results LINK Excel.Sheet.12 "C:UsersTyler-leaOneDriveDesktopUSQ SubjectsYEAR 3 - Trimesters 1,2 & 3Trimester 2Concepts in EndocrinologyResidential school activity 2 results.xlsx" "CORTISOL ELISA EXPERIMENT!R1C9:R13C11" a f 4 h * MERGEFORMAT

-5842002495550Fig.1 Standard Curve for Hair Cortisol levels in individual sample was measured using ELISA with absorbance at 655 nm and analysed using Excel. Hair samples followed standard protocols, and doubling dilutions (10, 5, 2.5, 1.25, 0.625, 0.313, 0.156 g/dL) were used with positive, negative, and individual student sample. Data are presented as mean SEM. Significant trend (r = 0.9953).

0Fig.1 Standard Curve for Hair Cortisol levels in individual sample was measured using ELISA with absorbance at 655 nm and analysed using Excel. Hair samples followed standard protocols, and doubling dilutions (10, 5, 2.5, 1.25, 0.625, 0.313, 0.156 g/dL) were used with positive, negative, and individual student sample. Data are presented as mean SEM. Significant trend (r = 0.9953).

38011102479675Fig.2 Individual hair cortisol levels were measured using ELISA with absorbance at 655 nm and analysed with Prism. Doubling dilutions (10, 5, 2.5, 1.25, 0.625, 0.313, 0.156 g/dL) were used with positive, negative, and individual student sample. Data are presented as mean SEM.

00Fig.2 Individual hair cortisol levels were measured using ELISA with absorbance at 655 nm and analysed with Prism. Doubling dilutions (10, 5, 2.5, 1.25, 0.625, 0.313, 0.156 g/dL) were used with positive, negative, and individual student sample. Data are presented as mean SEM.

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293370034290000-55245033591500

29972002417445Fig.4 RS01 class hair cortisol levels (n=22) were measured using ELISA with absorbance at 655 nm and analysed with Prism. Hair samples were prepared according to standard protocols, and doubling dilutions (10, 5, 2.5, 1.25, 0.625, 0.313, 0.156 g/dL) were used with positive, negative, and individual student samples. Data are presented as mean SEM.

00Fig.4 RS01 class hair cortisol levels (n=22) were measured using ELISA with absorbance at 655 nm and analysed with Prism. Hair samples were prepared according to standard protocols, and doubling dilutions (10, 5, 2.5, 1.25, 0.625, 0.313, 0.156 g/dL) were used with positive, negative, and individual student samples. Data are presented as mean SEM.

-6032502417445Fig.3 Standard Curve for Hair Cortisol levels in RS01 class (n=22) were measured using ELISA with absorbance at 655 nm and analyzed using Excel. Hair samples followed standard protocols, and doubling dilutions (10, 5, 2.5, 1.25, 0.625, 0.313, 0.156 g/dL) were used with positive, negative, and individual student samples. Data are presented as mean SEM. Significant trend (r = 0.9897).0Fig.3 Standard Curve for Hair Cortisol levels in RS01 class (n=22) were measured using ELISA with absorbance at 655 nm and analyzed using Excel. Hair samples followed standard protocols, and doubling dilutions (10, 5, 2.5, 1.25, 0.625, 0.313, 0.156 g/dL) were used with positive, negative, and individual student samples. Data are presented as mean SEM. Significant trend (r = 0.9897).

-6159502188845Fig.5 Standard Curve for Hair Cortisol levels in RS02 class (n=18) were measured using ELISA with absorbance at 655 nm and analysed using Excel. Hair samples followed standard protocols, and doubling dilutions (10, 5, 2.5, 1.25, 0.625, 0.313, 0.156 g/dL) were used with positive, negative, and individual student samples. Data are presented as mean SEM. Significant trend (r = 0.9984).

00Fig.5 Standard Curve for Hair Cortisol levels in RS02 class (n=18) were measured using ELISA with absorbance at 655 nm and analysed using Excel. Hair samples followed standard protocols, and doubling dilutions (10, 5, 2.5, 1.25, 0.625, 0.313, 0.156 g/dL) were used with positive, negative, and individual student samples. Data are presented as mean SEM. Significant trend (r = 0.9984).

30416502258696Fig.6 RS02 class hair cortisol levels (n=18) were measured using ELISA with absorbance at 655 nm and analysed with Prism. Hair samples were prepared according to standard protocols, and doubling dilutions (10, 5, 2.5, 1.25, 0.625, 0.313, 0.156 g/dL) were used with positive, negative, and individual student samples. Data are presented as mean SEM.

00Fig.6 RS02 class hair cortisol levels (n=18) were measured using ELISA with absorbance at 655 nm and analysed with Prism. Hair samples were prepared according to standard protocols, and doubling dilutions (10, 5, 2.5, 1.25, 0.625, 0.313, 0.156 g/dL) were used with positive, negative, and individual student samples. Data are presented as mean SEM.

-54610024574500389890027749500

Discussion

Relevant journal articles supporting the discussion section must have a publishing date no older than 2019. This section requires 10 journal articles.

Discussion points to mention: Context of findings and compare to relevant literature. What does pg/mg mean? Relevant literature relating the results, what is STD range? What syndromes and how do they relate to this study? If result as individual not expected correctly provide limitations and improvements on how to fix the experiment in future. Explain CV value etc. (Was above 20%) not acceptable. Main point is to explain why it went wrong and how to prevent and overcome the issue. If low value = syndrome or if high value = syndrome important to relating it to real world literature.

Results to include in discussion:

P value for individual - 0.5833 Not Significant

P value for RS01 - 0.8333 Not Significant

P value for RS02 - >0.9999 Not Significant

For Intra-assay CV: High CV= Not precise, Low CV = Precise CV equal to or above 20 is cut off point.

Intra-assay CV for individual - 4.33 (Accepted below 20)

Intra-assay CV for RS01 - 5.72 (Accepted below 20)

Intra-assay CV for RS02 - 12.35 (Accepted below 20)

Individual Hair cortisol conversion (pg cortisol/ mg hair) = 133.29 pg/mg

RS01 Hair cortisol group mean average conversion (pg cortisol/ mg hair) = 593.08 pg/mg

RS02 Hair cortisol group mean average (pg cortisol/ mg hair) = 167.39 pg/mg

Limitations discovered:

Washing wells incorrectly, spilling samples into other wells causing contamination

Pipetting skills inaccuracy: when preparing the plates in pairs (RS01 & RS02) there were two sets of different techniques (human error)

For some students there was not enough sample prepared therefore not all 50uL was added into the plate wells to begin with.

Papper towel when drying the Elisa plate may have been too wet and could contaminate the plate wells

Bubbles in wells, this affected the plate absorbance readings and could have affected the binding of the antigens

Using PBS may have affected the background signal as it can stick to the plates wells, the use of Distilled water may have been better?

Dye or bleaching hair could affect the samples readings.

References

Chu, B., Marwaha, K., Sanvictores, T., Awosika, A. O., & Ayers, D. (2024, May 7). Physiology, stress reaction. StatPearls - NCBI Bookshelf. https://www.ncbi.nlm.nih.gov/books/NBK541120/

Franklin, B. A., Rusia, A., Haskin-Popp, C., & Tawney, A. (2021). Chronic Stress, Exercise and Cardiovascular Disease: Placing the Benefits and Risks of Physical Activity into Perspective. International Journal of Environmental Research and Public Health/International Journal of Environmental Research and Public Health, 18(18), 9922. https://doi.org/10.3390/ijerph18189922

Igboanugo, S., OConnor, C., Zitoun, O. A., Ramezan, R., & Mielke, J. G. (2023). A systematic review of hair cortisol in healthy adults measured using immunoassays: Methodological considerations and proposed reference values for research. Psychophysiology, 61(1). https://doi.org/10.1111/psyp.14474

Kim, E. J., & Kim, J. J. (2023). Neurocognitive effects of stress: a metaparadigm perspective. Molecular Psychiatry, 28(7), 27502763. https://doi.org/10.1038/s41380-023-01986-4

Knezevic, E., Nenic, K., Milanovic, V., & Knezevic, N. N. (2023). The role of cortisol in chronic stress, neurodegenerative diseases, and psychological disorders. Cells, 12(23), 2726. https://doi.org/10.3390/cells12232726

M, S., S, M., Vadakkiniath, I. J., & A, G. (2023). Prevalence and correlates of stress, anxiety, and depression in patients with chronic diseases: a cross-sectional study. Middle East Current Psychiatry, 30(1). https://doi.org/10.1186/s43045-023-00340-2

Meyer, J., Novak, M., Hamel, A., & Rosenberg, K. (2014). Extraction and Analysis of Cortisol from Human and Monkey Hair. Journal of Visualized Experiments, 83. https://doi.org/10.3791/50882

Roberts, B. L., & Karatsoreos, I. N. (2021). Brainbody responses to chronic stress: a brief review. Faculty Reviews, 10. https://doi.org/10.12703/r/10-83

Russell, E., Koren, G., Rieder, M., & Van Uum, S. (2012). Hair cortisol as a biological marker of chronic stress: Current status, future directions and unanswered questions. Psychoneuroendocrinology, 37(5), 589601. https://doi.org/10.1016/j.psyneuen.2011.09.009