In this experiment, diurnal rhythm of cortisol in salivary samples was measured by using Competitive ELISA.

1. University of East London
School of Health, Sport and Bioscience
BSM006 2013-2014
Diurnal Rhythm of Cortisol
Practical write-up report
Student name:Nima Taefehshokr

3. Introduction:
In 1905 for the first time the term ‘Hormone’ was coined which it comes
from Greek and means ‘excite’. Adrenal glands consist of a medulla and
cortex, the adrenal cortex is responsible for steroid hormone’s synthesis.
So, two types of adrenal cortical steroid hormone’s are secreted (Harddie,
1992). The first types of corticoids are mineralocorticoids which
significantly act in maintaining blood pressure, balancing salt and water in
the body with retaining sodium and potassium excretion by kidneys. The
second kinds of corticoids are glucocorticoids which includes corticosterone
and cortisol that sometimes are called ‘Hydrocortison’ (Neave, 2008).
Figure 1. cortisol
Cortisol has a range of different physiological impacts. It plays an
important role in liposis, helping the CNS to respond to various stimuli,
breaking down the muscle protein and glycogen (Stanfield, 2005). Moreover,
this hormone can inhibit response of the immune system cells for this
reason it can be used for having pharmacological impact. So, cortisol has
been applied in relieving the symptoms of disease such as rheumatoid
arthritis and in treating some kinds of allergies (Harddie, 1992).
Cortisol level can be assayed using urine, blood, and saliva samples and
because of some drawbacks relating to blood and urine, salivary assessment
is the most preferred method. For instance, anxiety can be generated by
taking blood sample and this can cause in rising of cortisol level temporarily.
Urine sample collection over 24 hours needs repetition and sticking to
timetable is hard and can be even impossible for young children because
there is considerable delay between urine production and stimulus (Kimball

4. et al, 2007). On the other hand, salivary cortisol measurement takes the
advantage of a cheap, painless, simple procedure plus it can easily be
collected, stored and analyzed (Neave, 2008).
In regarding to assign the level of salivary cortisol two types of tests have
a considerable role in its measurement. Firstly, radioimmunoassay (RIA),
with the use of radioisotypes is used in this process. Secondly, is by the
Enzyme Linked Immunosorbent Assay (ELISA) which can be done with very
small amounts of saliva and without utilizing any radioisotypes (Kimball et al,
2007). As, with 361 molecular weights cortisol is regarded as a small
molecule, which can be antigenic by binding to protein carriers and as a
result antibodies can be raised against them. Thus, competitive ELISA is
used to detect very small antigens which provide fast and easy way that
ideally suits with the cortisol level measurement (Outram, 2014).
Figure 2. Competition ELISA
Furthermore, measuring cortisol level is the confirmed method in diagnosing
the abnormal cortisol proportion, so this can reflect adrenal cortex
activity. This abnormality can be in forms of hypocortisolism and

5. hypercortisolism which can lead to Addison’s and Cushing’s syndromes
respectively (Johnson, 2008).
Methods:
In this experiment competitive ELISA is used to determine the level of
cortisol in saliva. In this type of ELISA test competition happens between
an enzyme labeled antigen (conjugate) and unlabeled antigen (exists in
standards and samples) for a limited number of antibody binding sites which
has been attached to a plastic well.
In this process which was done in duplicate, standard cortisol from 0-300
nM and saliva samples taken at different times, 06:00, 07:00, 08:00,
10:00, 12:00, 16:00, 20:00,24:00 were provided in the laboratory.
Firstly, labeled cortisol (peroxidase cortisol conjugate) was added to the
standard and patient sample well strips (2×8 standard and 2×8 patient
sample coated with anti cortisol). Then,standard solutions and unknown
cortisol saliva samples were added to each microtiter wells of standard and
patient samples respectively. Secondly, well strips were incubated for 1
hour at room temperature. In the next step, for removing unbound
materials the wells were washed three times with washing buffer. After
decanting and washing steps enzyme substrate, tetra methyl benzidine
(TMB) was added and 20 minutes were allowed to develop color.
Subsequently, color development reaction was terminated by addition of
HCL as a stop solution which the color of the wells was changed from blue
to yellow. Finally, absorbance in each well was read at 450nm by a
microtiter plate reader.
Results:
Section 1:

6. The below table reflects cortisol standards absorbance readings and the
average of absorbances at 450nm with the concentration range of 0 to 300
nM which was used to draw the calibration curve.
Graph (1) shows important aspects of cortisol level which was the highest
one around 0.89 OD in 0.3 nM and then it declined gradually to it’s lowest
at about 0.36 OD in 120 nM .
Standard nM Absorbance
450
Absorbance
450
Mean
0 0.872 0.776 0.824
0.3 0.911 0.885 0.898
1 0.902 0.801 0.851
3 0.78 0.76 0.77
10 0.689 0.652 0.670
30 0.567 0.543 0.555
100 0.43 0.42 0.425
300 0.343 0.38 0.361
Table 1. Cortisol standards, absorbances and average of absorbances at 450nm.
Graph 1. Calibration Curve.
0.3615
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0.1 1 10 100 1000
Standard Curve
nM Cortisol
ODUnits
6
7
8
10
12
16
20
24
Average

7. Section 2:
In this section the table (2) indicates the duplex absorbance of the patient
sample in a range of different hours. Also the mean of the absorbances
was calculated and the level of cortisol was found on a various times during
a day by using the average in the standard curve.
Time Absorbance
450
Absorbance
450
Mean Cortisol Level
6 0.464 0.456 0.46 60
7 0.555 0.501 0.528 34
8 0.58 0.52 0.55 27
10 0.651 0.632 0.641 12
12 0.684 0.69 0.687 8
16 0.715 0.73 0.722 6
20 0.723 0.755 0.739 5
24 0.736 0.788 0.762 4
Table 2. time of samples, absorbance readings, absorbance averages and cortisol levels.
Graph 2. Diurnal rhythm of cortisol.
0
10
20
30
40
50
60
70
0 2 4 6 8 10
Diurnal rhythm of cortisol
Nmcortisol
24 hours

8. Discussion:
In this experiment level of the diurnal cortisol was shown in graph (2) which
showed high amount of secreted cortisol, 60nM at 6:00. Then, its secretion
declined and reached to 27nM after two hours. So, cortisol secretion
demonstrated a dramatic decrease between 6:00 and 12 which it accounted
for 60nM and 8nM respectively. Afterwards, cortisol level even fell into its
lowest, 4nM by 24.
According to the results of secreted cortisol, that’s clear that cortisol
rises significantly (50-160% in saliva) in the morning and drops during the
day. In the other words, cortisol synthesis follows a distinctive diurnal
pattern in which it’s in highest amount early in the morning and late at
night it reduces to the lowest level (Kimball et al, 2007). On the other
hand, because of a different life style sometimes cortisol secretion is not
based on the diurnal rhythm. Plus, stress is the first key factor in
stimulating cortisol to secrete, so harmful stimuli such as: strenuous
exercise, long term starvation, pain, infection, shock, burn, trauma and
surgery can play a crucial, considerable role in cortisol secretion (Stanfield,
2005).
Meanwhile, to cortisol secretion, Hypothalamic-Pituitary-Adrenal (HPA) axis
must be activated. So, mentioned stimuli, such as stress stimulate
hypothalamus to secrete corticotropic- releasing hormone (CRH) into the
hypophyseal portal system and this will be carried to the anterior pituitary.
Subsequently, CRH stimulates adrenocorticotropic hormone (ACTH) to be
produced from the anterior pituitary. At last, ACTH motivates adrenal
cortex to release cortisol and then circulating cortisol can have a negative
feedback and lead to inhibition of CRH and ACTH secretion (Neave, 2008).

9. Figure 3. Cortisol secretion pathway.
Furthermore, human body often secrete cortisol abnormally which can lead
to some serious conditions and it can be divided in two groups, called
hypercortisolism and hypocortisolism. Also, deficiency and excess of cortisol
production can be because of a defect either deriving from the adrenal
cortex (Primary disorder) or a deficiency in the release of the CRH or
ACTH hormones (Secondary disorder).In the case of hypercotisolism,
Cushing’s disease (Figure 4.) is the distinct example of it that can be
characterized with, obesity, moon face, buffalo hump (indicating adipocytes
synthesis in certain areas of the body and untypical fat distribution) ,easy
skin bruising, hirsutism and acne on face (Stanfield, 2005).

10. Figure 4. symptoms of cushing’s syndrome.
On the other hand, Addison’s disease (Figure 5.) is the typical case
representing hypocortisolism and signs includes, hypoglycemia, abnormal skin
pigmentation, weight loss, muscle weakness and fatigue and low blood
pressure (Johnson, 2008).
Figure 5. Symptoms of Addison’s disease.

11. References:
1. Neave, N. (2008). Hormones and behaviour. New York: Cambridge
University Press.
2. Hardie, D. (1992). Biochemical messengers. London: Cambridge
University Press.
3. Stanfield, C.L. (2005). Principles of human physiology. 4th
ed. Boston:
Benjamin cummings.
4. Johnson, B.R. (2008). Human physiology. Benjamin cummings.
5. Outram, S. (2014). Detection of the diurnal rhythm of cortisol secretion
lecture.
6. G.Kimball.J., M.Lynch.K., C.Stewart.K., E.Williams.N., A.Thomas.A.
and E.Atwood.K. (2007).Using salivary cortisol to measure the effects of a
Wilbarger protocol- based procedure on sympathetic arousal. The American
Journal of Occupational Therapy, 61 pp. 406-413.