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.
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 50ml 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
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.
Figure 2: ACTH stimulation test of two patients: the above figure shows the variance of ACTH stimulation test of patient A and B at 0, 30 and 60 minutes time interval. The cortisol level of patient A rises significantly within 30 minutes
Figure 3: Dexamethasone suppression test for two patients. Both patient cortisol concentration reduced from 0-9am to 2-9am different time intervals. However, Patient A shows considerable drop of cortisol concentration.
An increase of cortisol level, for instance, 7 ug/dL baseline to 10ug/dL, or attaining at least 18ug/dL level at 60 minutes post stimulation efficiently rules out primary adrenocortical insufficiency (AI) and proposes that adrenal suppression is negligible (Ceccato et al. 2014). An absent or blatant reaction proposes that some levels of secondary AI. If a substandard reaction is realized with a raised baseline ACTH phase, the patient has a primary AI or a kind of ACTH insensitivity (Ceccato et al. 2014).
Basically, the ACTH stimulation depends on negative feedback, where stimulus is utilised from the body natural regulation of itself to maintain ideals levels (Thompson et al. 2016). The ACTH stimulation levels test mainly the use of a synthetic form of ACTH, which is injected into the patients during the test. The projection is that the body will react to an additional of ACTH by generating cortisol from the adrenal cortex. After one hour, the levels of cortisol and a related hormone in the adrenal glands, aldosterone, are gauged in the serum (Thompson et al. 2016). The cortisol level ought to increase above the baseline which shows that the adrenal tissue is functional and responsive to ACTH. This is illustrated on the figure two, where the patient A shows an abrupt increase in the cortisol concentration (Thompson et al. 2016).
The ACTH stimulates the cortisol production, irrespective of the health of the pituitary glands. If suitable ACTH was provided and there was no increase in cortisol levels, the adrenal cortex is projected to be sources of disease (Ceccato et al. 2014). According to this experiment, patient B in figure 2 shows decrease and very low cortisol concentration. This implies that the patient could be suffering from adrenal defects. This result narrows down the differential diagnoses to syndrome such as Addison’s disease and adrenal insufficiency, as well as diseases linked with the lower cortisol levels (hypocortisolism) in the patient B (Frank et al. 2015).
On the contrary, if the levels of cortisol rise significantly above 20mcg/dl, the pituitary glands itself may have a disease such as hypopituitarism (low hormone secretion) (Ceccato et al. 2014). If the patient has come in with the low blood sugar and low blood pressure, there are symptoms of low cortisol. Thus, if their control spikes overly high the ACTH test, it is probably that the concerns are coming from the pituitary glands for further upstream at the hypothalamus (Ceccato et al. 2014). The above statement is well related to the patient A in figure 2, where the level of cortisol rises significantly within 30 minutes (Frank et al. 2015).
Elucidation of the outcomes of a low-dose dexamethasone suppression assessment must be centred on the laboratory's standard array for the dosage and preparation of dexamethasone directed (Frank et al. 2015). If the prescription of dexamethasone flops to sufficiently suppress flowing cortisol intensities in a patient with well-matched clinical indications, a finding of hyperadrenocorticism is established (Frank et al. 2015). If a plasma cortisol strength determined two to six hours after dexamethasone inoculation is suppressed routinely or near-routinely (to below 40 mmol/l), while the 8-hour trial displays to leak from cortisol suppression, then a finding of pituitary-dependent hyperadrenocorticism is directed (Frank et al. 2015). Therefore, in figure 3 above, both patient C and D, shows below 40mmol/L cortisol concentration. Thus, both patients can be diagnosed with the hyperadrenocorticism syndrome (Frank et al. 2015).
Normal people without the syndrome of the hypothalamus-pituitary-adrenal (HPA), at midnight they have low or unnoticeable cortisol level which generate up overnight to peak in the morning (Ceccato et al. 2014). Then, the level declines slowly through the day. Patients who are cortisol deficiency are well-known to suffer from adrenal insufficiency (AI). The condition generally presents deception and may be easily overlooked. In figure 1 above, the cycle correlate to the sequence of the normal individuals without HPA syndrome. The cycle shows a very high concentration of the cortisol in between 6 am and 9 am, and the processes reduce as the time progresses until evening. During the midnight the cycle is at lowest level in cortisol concentration (Thompson et al. 2016).
To conclude, Cosyntropin is synthetic ACTH-derived that is utilised in the assessment and analysis of patients with adrenocortical deficiency. Cosyntropin stimulation testing remains to be a keystone of diagnosing both the primary and secondary AI. While the low and high dose version has been presented to diagnosis AI, the low dose has been revealed to be more profound and precise.
Ceccato, F., Antonelli, G., Barbot, M., Zilio, M., Mazzai, L., Gatti, R., Zaninotto, M., Mantero, F., Boscaro, M., Plebani, M. and Scaroni, C., 2014. The diagnostic performance of urinary free cortisol is better than the cortisol: cortisone ratio in detecting de novo Cushing's syndrome: the use of a LC–MS/MS method in routine clinical practice. European journal of endocrinology, 171(1), pp.1-7. Retrieved from: https://www.eje-online.org/content/171/1/1.short, [Accessed on 17 November 2018].
Frank, N., Andrews, F., Durham, A., Kritchevsky, J., McFarlane, D. and Schott, H., 2015. Recommendations for the diagnosis and treatment of pituitary pars intermedia dysfunction (PPID). Equine Endocrinology Group (Internet), pp.4-5. Retrieved from: https://sites.tufts.edu/equineendogroup/files/2015/12/2015-10-16_EEG-2015-recommendations.pdf, [Accessed on 17 November 2018].
Thompson, S., Daly, S., Le Blanche, A., Abidi, M., Belkhiria, C., Driss, T. and de Marco, G., 2016. fMRI randomized study of mental and motor task performance and cortisol levels to potentiate cortisol as a new diagnostic biomarker. Journal of Neurology & Neuroscience, 7(2), p.92. Retrieved from: https://eprints.bournemouth.ac.uk/24458/1/2016-France-ST.pdf, [Accessed on 17 November 2018].