C700 Biochemistry

Question:

If you want to write a good report about a Scientific project of any kind, the format must be clear and concise.

The format for scientific reports is standardized. You need to be familiar with it.

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Here are some examples:

A title should be the first thing in a report.

A scientific paper typically has five sections.

Introduction: Give a brief description of the problem and the reason for it. Also, include a description of how the work was done.

Methods: This section should be described of what you did and not a series of instructions.

Results: These are the heart of a report.

You should discuss the key qualitative and quantitative findings in your work.

The data should be described and/or tabulated.

Answer:

Cortisol is a key steroid hormone that the adrenal gland releases. Like many biological processes in our body, it also has a circadian rhythm.

An investigative perspective may be crucial in determining the adrenal gland’s response to exogenous adrenocorticotropic hormonal (ACTH).

An artificial ACTH analogue cosyntropin, which is made up of 24 Nterminal amino acids of an instinctive peptide and is used for research purposes, is, in general, useful (Ceccato et al.

The amount of response depends not only on the functional integrity of a gland, but also on the scope before stimulation.

To diagnose adrenocortical deficiency, the above can be used to measure cortisol levels and to determine Congenital adrenal hyperplasia. (Frank and al.

A few trials are required to establish basal plasma cortisol levels. After that, intravenous cosyntropin of approximately 25 ug is administered.

After ACTH administration, the plasma is tested at intervals of 30-60 minutes.

After stimulation, plasma cortisol levels will increase beyond 15ug/L.

Dexamethasone (Ceccato, et al.) is the glucocorticoid choice. It is 25 times stronger that cortisol.

Dexamethasone does not suppress the pituitary release of endogenous ACTH. It doesn’t interfere with the adrenal reaction to exogenous ACTH.

Cushing syndrome patient, effective cortisol suppression doesn’t happen with glucocorticoid treatment because of the continuous production of cortisol.

Due to its strength as a synthetic glucocorticoid it is used in suppression experiments. The small quantity required to stop ACTH from being produced does not restrict the use of steroids assays.

Cushing’s condition can be monitored by a small dose.

The untrue positives could occur when the limit value of 70-200nmol/L is used (Thompson, et al.

Unreacted substrate should appear colorless or very pale yellow.

Peroxidase can react substrate with peroxidase to produce a soluble yellow or blue reaction product.

A suitable stop solution can be used to block the reaction, producing a soluble yellow/blue reaction product. It all depends on the stop substance.

Methods

25uL Cortisol standards, quality control samples, and patient’s samples were pipetted. 200ul of the conjugate Cortisol–Horseperoxidase was then added to each well.

Plaque was covered with cling film and placed on a plate shaker. Incubation took one hour. Samples were washed four more times in wash buffer in the washer machine. The plates were then tapped vigorously on a paper filter until dry.

In all wells, 200 uL substrate TMB solution were added.

After sealing the plate with clingfilm, incubation in dark was done to encourage colour growth. The result was darkish-blue.

To stop the reaction and further colour growth, incubate for approximately 30 minutes. After that 50ml (2M) sulphuric acids (2M), was added to all.

After addition, the wells began to turn yellow.

The plates were read at 450nm by a plate reader.

Results and Discussion

Figure 1: Circadian cortisol rhythm: The diagram above shows the cortisol concentration (nmol/L), at different times during the day.

It is clear that the circadian rhythm of cortisol rises in the morning. This increases quickly as it approaches 9am.

After that, cortisol levels decrease until midnight.

The diagnosis should be done between 6am – 9am.

Figure 2: ACTH stimulation testing of two patients. This figure shows ACTH stimulation test variances of patient A and patient B at 0, 30 or 60 minutes.

Within 30 minutes, patient A’s cortisol level increases significantly

Figure 3. Dexamethasone suppression tests for two patients.

Both patients saw their cortisol concentrations drop from 0-9am down to 2-9am at different intervals.

However, Patient A shows a substantial drop in cortisol.

For example, a cortisol level increase of 7 ug/dL to 10 ug/dL after stimulation or a minimum 18 ug/dL level 60 minutes later, effectively excludes primary adrenocortical impairment (AI).

If there is no obvious reaction, it suggests that the patient has secondary AI.

If a substandard response is observed with a higher baseline ACTH phase, it could indicate that the patient has primary AI. (Ceccato et. al.

ACTH stimulation is basically based on negative feedback. The body naturally regulates itself to maintain ideal levels of stimulation (Thompson, et al.

ACTH stimulation levels measure primarily the use of a synthetic ACTH form, which is administered to patients during the test.

The expectation is that the body will produce cortisol by reacting to ACTH.

The serum measures the levels cortisol and aldosterone in the adrenal glands after an hour (Thompson, et al.

The baseline cortisol level should rise above this, which indicates that adrenal tissue functions and is responsive to ACTH.

The figure 2 shows the same thing. A patient with a sudden increase in cortisol levels (Thompson, et al.

ACTH stimulates cortisol levels regardless of whether or not the pituitary hormones are healthy.

The adrenal cortex can be a source for disease if it is not provided with adequate ACTH (Ceccato and al.

Figure 2: Patient B shows a decrease in cortisol concentration.

This suggests that the patient might have adrenal defects.

This results narrows down possible diagnoses such as Addison’s disease or adrenal insufficiency.

However, hypopituitarism (low hormone production) may occur if cortisol levels rise significantly beyond 20mcg/dl (Ceccato, et al.

Low cortisol is a sign of low blood sugar and/or blood pressure if the patient presents with these symptoms.

Therefore, if the ACTH test shows that their blood sugar and blood pressure are high, then it is likely that they are experiencing concerns from the pituitary glands.

The above statement is closely related to A in figure 2. In this case, the cortisol level rises rapidly within 30 minutes (Franke et al.

Low-dose dexamethasone suppression assessment results must be based on the standard array used by the laboratory to determine the dose and preparation of dexamethasone (Frank et.al.

Frank et. al. found that if the prescribing of dexamethasone fails to suppress cortisol levels in a patient with well-matched clinical indications it is hyperadrenocorticism.

Frank et. al. reported that a plasma cortisol concentration of between 40 and 60 mmol/l can be determined after dexamethasone administration. If the plasma cortisol concentration is lower than this, or is near-routinely suppressed (to below 40mmol/l), then it is possible to determine if there is hyperadrenocorticism in the pituitary.

In figure 3, both patients C, and D have below 40mmol/L cortisol levels.

Frank et al. have diagnosed both patients with hyperadrenocorticism syndrome.

The levels gradually drop throughout the day.

People with cortisol deficiencies are more likely to be suffering from adrenal insufficiency.

It is easy to overlook this condition as it can be deceiving.

Figure 1 shows the same cycle as the normal sequence for individuals with HPA syndrome.

The cortisol levels are high in the morning between 6 and 9, and then decrease as the day progresses to evening.

The cycle’s lowest cortisol concentration is between midnight and 3:00 a.m. (Thompson, et al.

Cosyntropin (synthetic ACTH-derived) is used to assess and analyze patients with adrenocortical dysfunction.

Cosyntropin stimulation testing is still a critical tool for diagnosing primary and secondary AI.

Although both high and low dose versions have been used to diagnose AI, it has been demonstrated that the low dose is more effective and more precise.

In routine clinical practice, a LC-MS/MS test is used to determine if urinary free cortisol has a higher diagnostic accuracy than cortisol:cortisone ratio.

European journal on endocrinology, 171(1). pp.1-7.

Frank, N. Andrews F., Durham A., Kritchevsky J., McFarlane D. and Schott H.

Recommendations regarding the diagnosis and treatment for pituitary intermedia dysfunction (PPID).

Equine Endocrinology Group (Internet), pages 4-5.

Thompson, S. and Daly. S., Le Blanche. A., Abidi. M., Belkhiria. C. Driss. T., and de Marco. G. 2016. fMRI-randomized study of motor and mental task performance and cortisol level to potentiate the use of cortisol in diagnostic biomarkers.

Journal of Neurology & Neuroscience 7, 2: p.92.

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