Cortisol as biomarker for diagnostics PDF

Cortisol as biomarker for diagnostics
Introduction
Cortisol is an important steroid hormone released by the adrenal gland and just like numerous
biological process in human body, it has a circadian rhythm. The reaction of an adrenal gland
to exogenous adrenocorticotropic hormone (ACTH) may be important from an investigative
viewpoint. Generally, an artificial ACTH analogue cosyntropin, comprises of a 24 N-terminal
amino acids of an instinctive peptide, it is used for research purposes (Ceccato et al. 2014).
The level of reaction is not merely on the functional integrity of the gland but also the scope
before stimulation. The above is utilised to diagnose adrenocortical insufficiency by
measuring cortisol levels to detect Congenital adrenal hyperplasia (CAH) and androgen
levels (Frank et al. 2015). For the quick ACTH test, one or two trials are performed to
establish the basal levels of plasma cortisol, followed by an intravenous dose of about 25ug
cosyntropin. The plasma is tested at 30 and 60 minutes time intervals, after the administration
of ACTH. With the regular reaction to stimulation, the plasma cortisol will go beyond 15ug/L
and will show an increase of 5-7ug/dl or even more (Ceccato et al. 2014). Dexamethasone is
a glucocorticoid of choice and it is 25 times stronger than cortisol (Ceccato et al. 2014). Even
though dexamethasone suppresses the pituitary discharge of endogenous ACTH, it does not
inhibit the reaction of an adrenal to exogenous ACTH, thus, will not hinder the test outcomes
(Thompson et al. 2016).
The single-dose-dexamethasone test is used in diagnosing patients alleged of having Cushing
syndrome. Patient with the Cushing disease, effective cortisol suppression discharge does not
happen with glucocorticoid direction because of the steady production of cortisol (Ceccato et
al. 2014). Dexamethasone, being a strong synthetic glucocorticoid, it is utilised in
suppression experiments because the trivial quantity needed to quash ACTH, does not restrict
the steroids assays. A small single dose is utilised in an overnight suppression investigations

to monitor Cushing’s condition. But, the untrue negatives may happen when the limit values
of 70-200nmol/L are used (Thompson et al. 2016). TMB ELISA Substrate detects
horseradish peroxidase (HRP) action and encompasses 3, 3’, 5, 5’-tetramethylbenzidine in a
slightly acidic buffer. Unreacted substrate should be colorless or very light yellow in
appearance. When substrate method is reacted with peroxidase, a soluble blue reaction
product is attained. The reaction can be blocked using suitable stop solution, generating a
soluble yellow or blue reaction product, reliant upon the stop substance utilised.
Methods
25uL of Cortisol standards, quality controls, patient’s samples and cortisol standard samples
was pipetted and 200 ul of the conjugate Cortisol-Horse peroxidase conjugate was added in
all wells. Plate with cling film was sealed and incubated on a plate shaker Incubation in one
hour comprised washing samples four times with wash buffer in the washer machine and
tapped strongly on a paper filter until the plate was dried up fully. 200uL of substrate TMB
solution was added in all wells. TMB ELISA Substrate being very sensitive, identifies
horseradish peroxidase (HRP) action and comprises 3, 3, 5, 5’-tetramethylbenzidine in a
slightly acidic buffer. Plate with cling film was sealed and incubated in the dark for colour
growth which turned into a darkish blue. Incubate for around 30 minutes was done and
afterwards 50l sulphuric acid (2M) was added to all well so as to stop the reaction and hence
more colour growth. Upon addition, the wells turned yellow. Plates were observed on a plate
reader, at 450nm.

Results and discussion
6am 30
9am 70
1pm 30
4pm 20
8pm 8
12am 3
0 1 2 3 4 5 6 7
0
10
20
30
40
50
60
70
80
1; 30
2; 70
3; 30
4; 20
5; 8
6; 3
Circadian Rhythm of Cortisol)
cortisol (nmol/L)
Time
Cortisol (nmol/L)
Figure 1: Circadian rhythm of cortisol: the above diagram shows the concentration of
cortisol (nmol/L) at different time of the day. It is evident from the figure that circadian
rhythm of cortisol is high in the morning, which increases rapidly as it approaches 9am.
Afterwards, the cortisol level reduces until the midnight. Therefore, it is recommended
for the diagnosis to be performed between 6am to 9am.