Diurnal Rhythm of Cortisol - Essay Example

Cortisol is a steroid hormone made by the adrenal cortex following the production of adrenocorticotropic hormone (ACTH) by the anterior pituitary gland (Kelling 2008). It is often produced when the body encounters stress, an infection or an injury. Cortisol plays several vital roles in the body such as glucose metabolism and absorption of calcium. It also plays a role in the immune system particularly in anti-inflammatory responses (Hermann et al. 2006). Currently, salivary cortisol is usually used as an indicator of physiological stress as well as other mental and physical conditions (Hellhammer, Wust, & Kudielka 2009). It is thought that the concentrations of cortisol in the saliva offer a dependable estimate of the hypothalamus-pituitary-adrenal axis (HPAA) acclimatization to stress (Haussmann, Vleck, & Farrar 2007). It is, therefore, necessary to measure cortisol in a patient’s sample to assess the functioning of the adrenal glands in addition to the pituitary gland that is responsible for the initiation of the production of cortisol. It is also important in the detection and management of certain conditions such as Cushing’s disease and Addison’s disease (Jerjes et al. 2005). The hormone can be detected in blood, urine or saliva. Salivary cortisol is considered the most non-invasive way of determining cortisol levels. The most reliable way of measuring the amount of cortisol is through a technique known as enzyme-linked immunosorbent assay (ELISA). There are three forms of ELISA namely competitive ELISA, sandwich and indirect ELISA. Competitive (competition) ELISA is the most dependable way of measuring salivary cortisol because it allows for the detection of active cortisol that can compete with labelled cortisol. Figure 1: Competition ELISA assay (Principle of ELISA n.d.). In this assay, an antibody specific to the antigen being tested is immobilized on a well or a microtitre plate. A mixture containing a known concentration of the labelled antigen and the test sample is then added to the well. The labelled antigen and unlabeled antigen compete for the binding site on the immobilized antibody (Parija 2009). Any unbound antigen is washed after which a substrate is added. The substrate reacts with the enzyme to form a coloured substance that is then detected and the intensity of the colour is measured using an ELISA reader. The intensity of the colour is indirectly related to the concentration of the antigen in the test sample. Method Competitive ELISA was used to determine the quantity of cortisol in the salivary samples. The wells were coated with anti-cortisol antibody at a certain concentration such that a given quantity of antigen bound to the antibodies. A mixture of a known amount of cortisol antigen labelled with peroxidase and the patient’s saliva was then added to the well. The labelled antigen competed with the unlabelled antigen to bind to the immobilized anti-cortisol antibodies in the well. The unbound antigen was then washed to enable only the bound antigen to be quantified. An enzyme substrate was added to the wells to be acted upon by the enzyme on the labelled antigens. The plates were then incubated for about twenty minutes. This allowed for enzymatic reaction and the subsequent colour reaction. Thereafter, the stop solution was added to terminate the enzyme reaction. The absorbance of the plates was then read at 450 nanometres in an ELISA plate reader within twenty minutes of adding the stop solution. Intense colour of the wells indicated a low concentration of cortisol in the sample. The assay was initially run with an array of known cortisol concentrations (standards). A calibration curve was made by plotting the absorbance against the concentrations of the standards. The absorbance obtained from the samples was then used to determine the concentration of cortisol in the patient samples using the calibration curve. Results Table 1 below shows the absorbance recorded for the cortisol standards at 450 nm. The averages of the duplicate measurements were found and were used to obtain the calibration curve. Table 1: Standard Cortisol absorbance at 450nm Standard Cortisol (mM) Absorbance at 450nm Average Absorbance at 450nm 0 0.872 0.776 0.824 0.3 0.911 0.885 0.898 1 0.902 0.801 0.852 3 0.780 0.760 0.770 10 0.689 0.652 0.671 30 0.567 0.543 0.555 100 0.430 0.420 0.425 300 0.343 0.380 0.362 Figure 2: The calibration curve for cortisol The calibration curve in figure 2 above was obtained by plotting the average absorbance recorded at 450 nm against the known concentrations of the standards. Table 2: Table of salivary samples absorbance and cortisol concentration Samples (Salivary) at hours taken Absorbance at 450nm Average Absorbance at 450nm Cortisol Concentration (nM) 6 0.464 0.456 0.460 60 7 0.555 0.501 0.528 19 8 0.580 0.520 0.550 15 10 0.651 0.632 0.642 11 12 0.684 0.690 0.687 7 16 0.715 0.730 0.723 5 20 0.723 0.755 0.739 4.8 24 0.736 0.788 0.762 3.5 Table 2 above shows the recorded absorbance recorded for the patient samples. The average of the absorbance was recorded and used to determine the concentration of cortisol in the samples by extrapolation on the standard curve. Figure 3: A graph of patient diurnal rhythm of cortisol Figure 3 above shows the patient’s diurnal rhythm of cortisol. It was realized that the concentration decreased over the 24-hour period. The highest concentration of cortisol was obtained the initial sample (at six hours), whereas the lowest concentration was realized at 24 hours. Discussion It was observed that high levels of cortisol were expressed in the early part of the day (in the first 6-7 hours). On the contrary, the levels of cortisol decreased as the day progressed, and the lowest levels were observed at 24 hours. The results of this study were in agreement with several other studies carried out on the diurnal rhythm of cortisol (Hellhammer, Wust, & Kudielka 2009). These studies have shown that cortisol levels are often high in the morning and decrease throughout the day reaching the lowest concentrations in the early phases of sleep (Hermann et al. 2006; Adam n.d.). An individual benefits from the changed levels of cortisol during the day because it allows healthy growth and development (Hellhammer, Wust, & Kudielka 2009). The expression of cortisol is determined by the physiological and physical state of an individual. Emotional instability such as during stress causes an elevated expression of cortisol (Kunz-Ebrecht et al. 2003). A number of studies show that individuals with pressing issues on their minds produced more cortisol than those with relaxed minds (Haussmann, Vleck, & Farrar 2007). In addition, age is thought to affect the expression of cortisol and the resulting diurnal pattern. For example, a study by revealed that infants below the age of six moths did not have any variation in their diurnal rhythm of cortisol (Hindmarsh et al. 1989). During stress, high levels of cortisol direct resources (nutrients and energy) towards handling the stress leaving little resources for growth. Therefore, it is necessary that these levels change to create a balance in the use of energy and other nutrients. References Adam, E. K. n.d. Diurnal Cortisol rhythms: social determinants and role as a risk, state or scar marker for major depressive disorder in youth, viewed 17 March 2014, . Haussmann, F. M., Vleck, C. M., & Farrar, E. S 2007, “A laboratory exercise to illustrate increased salivary cortisol in response to three stressful conditions using competitive ELISA,” Advances in Physiology Education, vol.31 no. 2007, pp. 110–115. Hellhammer, D. H., Wust, S., Kudielka, B. M 2009, “Salivary cortisol as a biomarker in stress research,” Psychoneuroendocrinology vol.34 no.2009, pp. 163—171. Hermann, C., Aulock, S., Dehus, O., Keller, M., Okigami, H., Gantner, F., Albrecht Wendel, A., & Hartung, T 2006, “Endogenous cortisol determines the circadian rhythm of lipopolysaccharide- but not lipoteichoic acid-inducible cytokine release,” European Journal of Immunology, vol.36 no.2, pp 371–379. Hindmarsh, K. W., Tan, L., Sankaran, K., & Laxdal, V. A 1989, “Diurnal rhythms of cortisol, ACTH, and p-endorphin levels in neonates and adults,” Western Journal of Medicine, vol.151 no. 1989, pp. 153-156. Jerjes, W. K., Peters, T. J., Taylor, N. F., Wood, P. J., Simon Wessely, S., Cleare, A. J 2005, “Diurnal excretion of urinary cortisol, cortisone, and cortisol metabolites in chronic fatigue syndrome,” Journal of Psychosomatic Research vol.60 no.2006, pp. 145–153. Kelling, S. A 2008, An examination of salivary cortisol concentrations and behavior in three captive African elephants (Loxodonta africana) at Zoo Atlanta, ProQuest LLC, Ann Arbor, MI. Kunz-Ebrecht, S. R., Mohamed-Ali, V., Feldman, P. J., Kirschbaum, C., & Steptoe, R 2003, “Cortisol responses to mild psychological stress are inversely associated with proinflammatory cytokines,” Brain, Behavior, and Immunity, vol.17 no.2003, pp. 373–383. Parija, S. C 2009, Textbook of microbiology & immunology, Elsevier, Haryana, India. Principle of ELISA n.d., viewed 18 March 2014 . Read More