Adrenocorticotropic Hormone (ACTH)

Adopt a Hormone

Alexandra Barbera
SBL 322 01 Endocrinology
Dr. Bassett
April 27, 2017
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Hormones are an important part of how the body functions. From simple actions of

digestion to more complicated aspects of homeostasis, hormones have a great impact on keeping

our bodies working correctly. When hormones are off-balance disease, illness or even death can

occur. Our bodies usually work in feedback systems to regulate most hormones, but when there

is a tumor or mutation, the amount of hormone in the body can be affected. Each hormone in the

body has a specific action and can work in a system with other hormones to produce an action(s).

Adrenocorticotropic hormone, or corticotropin, is a hormone produced in the adrenal cortex that

has a big impact on the human body.

In class, we discussed many topics dealing with adrenocorticotropic hormone, ACTH,

and its related hormones. ACTH is a tropic hormone, which means it is a hormone which

stimulates another hormone to do something, and in this case works with cortisol. The process of

making adrenocorticotropic hormone starts in the hypothalamus. The paraventricular nucleus

(PVN) in the hypothalamus produces Corticotropin-releasing hormone (CRH) that travels to the

anterior pituitary to stimulate the release of ACTH by corticotrophs (Topic 8, Objective 1).

Corticotropin, another name for ACTH, is then released into the blood stream and travels to the

zona fasciculate of the adrenal cortex to stimulate the secretion of cortisol, a glucocorticoid,

along with the release of mineralocorticoids and androgens (Topic 8, Objective 1). ACTH not

only produces cortisol in the bundle like zona fasciculate, but also works on the zona reticularis

with luteinizing hormone and hCG to produce DHEA, DHEA-S and androstenedione (Topic 15,

Objective 2). Cortisol works in a negative feedback system in which elevated levels of the

hormone results in the inhibition of ACTH by corticotrophs and CRH by hypothalamic

neurosecretory cells (Topic 8, Objective 1). Corticotrophs are a type of hormone producing cell

that are present in the pars distalis of the adenohypophysis that produces ACTH (Topic 8,
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Objective 3). Adrenocorticotropic hormone is a category II tropic hormone, meaning it is a

proopiomelanocortin (POMC) derivative (Topic 9, Objective 2). In corticotrophs, prohormone

convertase 1 cleaves POMC into three smaller fragments including ACTH, -lipotropin and a

16K inactive fragment (Topic 9, Objective 2). ACTH can be cleaved further to produce -

melanocyte stimulating hormone and CLIP, corticotropin-like intermediate peptide by PC2

(Topic 9, Objective 2).

Adrenocorticotropic hormone does not exhibit continuous secretion, but follows a

circadian rhythm with a peak of ACTH production in the morning around 6am and a nadir after

midnight (Topic 10, Objective 5). Plasma cortisol follows the diurnal pattern of plasma

corticotropin with cortisol trailing the rhythm of ACTH production (Topic 15, Objective 3).

ACTH release is regulated not only by CRH, but also by arginine vasopressin and negative

feedback system of glucocorticoids, most often, cortisol (Topic 10, Objective 5). ACTH can be

blocked by actions of the pineal gland as well. Melatonin from the pineal gland blocks the

secretion of hypothalamic-releasing hormones, such as CRH, which would block ACTH

formation (Topic 11, Objective 5). Stress is the major factor in the stimulation of CRH to start

the hypothalamic-pituitary-adrenal axis that produces cortisol. This stress is chronic stress as

acute stress is dealt with by acts of norepinephrine and epinephrine (Topic 15, Objective 3). The

response to stress by ACTH and cortisol is lipolysis, gluconeogenesis, protein catabolism,

sensitized blood vessels, and reduced inflammation as well as changes in body weight,

suppression of reproduction, elevated glucocorticoid levels and possible immune suppression

(Topic 15, Objective 4). The PowerPoints also talks about work and cortisol levels. While there

was no difference in cortisol levels after work, during work, men with a lower pay grade had

elevated cortisol levels throughout the day and cortisol levels were lower on the weekends in
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male and females (Topic 15, Objective 4). Changes in fecal glucocorticoid content reflects the

response to ACTH, and this was demonstrated in aggression in both male and female hyenas.

The more aggression presented, the more glucocorticoids were found in their fecal matter (Topic

15, Objective 3).

Environmental factors also play a role in the regulation of adrenocorticotropic hormones.

The herbicide, atrazine, has no effect on ACTH or cortisol release itself; however, atrazines

metabolite, deisopropyl-atrazine, doubled the production of ACTH, and cortisol passively (Topic

16, Objective 1). In licorice and pseudohyperaldosteronism there is a chemical, glycyrrhizinic

acid which elevates cortisol levels through the inhibition of the isoform 11-HSD 2, which

normally deactivates cortisol by converting it to cortisone (Topic 16, Objective 1). The resulting

build up of cortisol leads to an aldosterone-like effect involving sodium and water retention in

the body (Topic 16, Objective 1).

The tropic-like hormone that is most comparable to that of adrenocorticotropic

hormone is that of chorionic corticotropin (Topic 9, Objective 1). The fetal hypothalamus

matures to the point where it can release regulatory levels of CRH and the adenohypophysis

corticotrophs mature to respond to the CRH and release ACTH where the adrenal cortex matures

to release cortisol and DHEA (Topic 18, Objective 3). Maternal stress causes the placental

release of CRH which in turn stimulates estrogen synthesis through the release of ACTH

releasing DHEA, a precursor for estriol, by the placenta to initiate contraction of the uterus

(Topic 18, Objective 3).

Malfunction of adrenocorticotropic hormone occur and there are diseases and conditions

associated with the hormone. There are sites, or ectopic nodules, throughout the body that

contain adrenal tissues other than the established adrenal glands (Topic 15, Objective 1). One
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abnormality from these ectopic sites is Cushings syndrome. Cushings syndrome is due to

hypersecretion of ACTH by non-pituitary tumors or of cortisol by the adrenal gland or other

tissues in the body and its cause is unknown, but is more prevalent in women (Topic 16,

Objective 2). The negative feedback system of cortisol on ACTH is responsible for the adrenal

gland atrophy and is treated by cortisol-suppressing drugs with possible surgery (Topic 16,

Objective 2). Another hypersecreting condition is that of Cushings disease. Cushings disease is

different from the syndrome in the aspect that the cause is still unknown, but an adenoma tumor

is present and there is a hypersecretion of ACTH from corticotrope cells in the pars distalis and

is more common in pregnant women (Topic 16, Objective 2). The treatment for Cushings

disease is commonly a surgery pioneered by Cushing himself, which is a transsphenoidal

adenomectomy, the removal of the pituitary though the sphenoid bone (Topic 16, Objective 2).

There are also conditions where there is hyposecretion, including Addisons Disease and

Congenital Adrenal Hyperplasia. Addisons disease is 70% idiopathic with adrenal

glucocorticoid and mineralocorticoid release is put to a stop resulting in an increased sodium

excretion and decreased potassium excretion with decreased blood volume (Topic 16, Objective

3). There is also an impaired glucose and lipid catabolism with an increase ACTH release which

cause a darker skin pigmentation due to in increase in -lipotropin stimulating melanocytes

(Topic 16, Objective 3). Congenital Adrenal Hyperplasia is an autosomal recessive disorder of

steroidogenic enzymes which result in hyponatremia, hypovolemia and a build up of unused

precursors that increase androgen activity (Topic 16, Objective 3). Treatment includes a drug

therapy that brings glucocorticoid and mineralocorticoid levels close to that of physiological and

androgen suppressants in females (Topic 16, Objective 3).

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Historically, there have been many important contributions and milestones in the

discovery and the understanding of the physiology of adrenocorticotropic hormone. The first use

of the word hormone was not until June of 1905 when Ernest Sterling used it in one Croonian

Lecture and defined the word from Greek meaning of to arouse or excite, (Tata 2005). He

described hormones as the chemical messengers which speeding from cell to cell along the

bloodstream, may coordinate the activities and growth of different parts of the body. (Tata,

2005). Adrenocorticotropic hormone is a tropic hormone that in turn regulates other hormones

and regulates stress and other factors in the body. These discoveries of corticotropin, or ACTH,

came from all over the world and greatly impacted the knowledge we have today of the tropic

hormone.

In 1930, the discovery lead by Smith was that of adrenocorticotropic hormone was a

factor produced by the pituitary that maintained the weight of the adrenal cortex (Harno, 2016).

Other contributions were made in the 30s by different scientists that investigated the properties

of the hormone. In 1933, research groups were lead by the Canadian biochemist James Collip

along with American biologist Herbert Evans and Argentine physiologist Bermado Houssay

(Medical Discoveries). These men used pituitary extracts in order to stimulate the center, or zona

fasciculate of the adrenal cortex (Medical Discoveries). Adrenocorticotropic hormone was then

not isolated until 1943 by American biochemist Choh Hao Li, and several other scientists

(Medical Discoveries). Adrenocorticotropic hormone today is often prescribed to reduce

inflammation in rheumatoid arthritis, ulcerative colitis and forms of hepatitis but its use was first

studied by two American medical researchers in search of an effective treatment for arthritis

(Medical Discoveries). Philip Hench and Edward Kendall headed the first program during World

War II to mass produce ACTH for medical use and in 1948-9 Hench and another colleague were
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the first to successively use the hormone in arthritis patients (Medical Discoveries). Hench and

Kendall won and received the 1950 Nobel Peace Prize in physiology for their achievement and

discovery (Medical Discovery).

Adrenocorticotropic hormones primary structure was first expressed in 1954 (Harno,

2016). In 1964, -lipotropin was isolated from ACTH as a pituitary hormone and in 1976 the

peptide -endorphin was isolated exhibiting opioid activity (Harno, 2016). High-molecular-

weight forms of ACTH were identified in 1973 in both human plasma and mouse pituitary cells

as well as human tumors which lead to predictions of a precursor for ACTH (Harno, 2016). It

was not until 1978 that there was proof that proopiomelanocortin, or POMC, was the common

precursor of ACTH, lipotrpon and endorphin and its sequence was determined in 1979 (Harno,

2016). Two years later, in 1981 the hypothalamic hormone, corticotropin-releasing hormone,

was isolated and its sequence was determined (Harno, 2016). Research in the 1980s showed that

stimulation with ACTH increases the amount of of receptor sites at the adrenal cell membrane

(Fridmanis, 2017). In 1992, the cloning of ACTH receptors was successful and in 1998 it was

found that inherited mutations in POMC and prohormone convertase 1 was associated with

early-onset obesity as well as adrenal insufficiency and the red pigmentation in hair (Harno,

2016). In more recent years, the mechanism of glucocorticoids regulation of POMC was

identified in 2005 and in 2007, the expression of the hypothalamic-pituitary-adrenal axis

equivalent was found in the skin (Harno, 2016). Also in 2005, two adrenocorticotropic hormone

receptors and four molecules of melanocortin receptor accessory protein were discovered

(Fridmanis, 2017). In 2011, the epigenetic control of POMC was found to have a role from

prenatal programming (Harno, 2016).

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Overall, the history and knowledge of the hormone we know today as either

adrenocorticotropic hormone or corticotropin had many contributors from all over the world and

the knowledge is continuing to grow. The dates and discoveries listed above helped to create a

better understanding of corticotropin itself as well as the related hormones and conditions that

goes along with it.

Adrenocorticotropic exhibits many characteristics involving its physiology, mechanisms

of action and pathophysiology. Adrenocorticotropic hormone is a polypeptide hormone

composed of 39 amino acids organized in a specific primary structure (Adrenocorticotropic

Hormone, n.d.). The chemical structure of ACTH is as follows: [Ser-Tyr-Ser-Met-Glu-His-Phe-

Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro-Asp-Gly-Glu-Ala-

Glu-Asp-Ser-Ala-Glu-Ala-Phe-Pro0Leu-Glu-Phe] with the N-terminal 24 amino acid segment

identical in all species (Thomas,1986). Its molecular formula is C207H308N56O58S with a

molecular weight of 4542.1 g/mol and its structure is shown in Figure 1 (Corticotropin, n.d.).

Amino acids 1-23 have full biological activity at the MC2R while 1-19 only have 80% activity

and 1-16 with very little activity (Carr, 2013). All ACTH fragments containing 1-13 amino acids

also have -MSH activity due to its messaging sequence His-Phe-Arg-Trp (Carr, 2013).

Corticotropin-like intermediate peptide (CLIP) is 22-amino acids and is the amino acids 18-39 of

ACTH (Thomas, 1986). The 1-32 sequence is the most potent in stimulating steroidogenesis with

6-24 showing some steroidogenic activity (Harno, 2016). The major role of adrenocorticotropic

hormone is to stimulate steroidogenesis in the adrenal cortex to release cortisol in humans

(Harno, 2016). ACTH is secreted from corticotropic cells of the pars distalis, anterior pituitary,

into the bloodstream in order to reach the adrenal in response to stress and CRH (Fridmanis,
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2017). Normal values of ACTH in the human body range from 9 to 52 pg/mL with the blood test

being the most accurate in the morning (Adrenocorticotropic Hormone, n.d.).

Figure 1, ACTH Structure (Corticotropin, n.d.)

Secretion of ACTH is influenced by stress, but is also influenced by the circadian rhythm.

As already mentioned, adrenocorticotropic hormone follows a circadian rhythm with its highest

levels in the morning and its lowest levels at night. It is secreted in a pulsile release process with

the amplitude, not the frequency, controlling the circadian rhythm with the rhythm itself varying

with different lifestyles (Harno, 2016). There is about 12-30 pulses of ACTH release per day and

with a half life of typically 14-35 minutes (Harno, 2016). Another influence of ACTH release

includes other hormones. Corticotropin releasing hormone is the most influential along with the

nonapeptide, arginine vasopressin, AVP (Carr, 2013). Corticotropin-releasing hormone

stimulates the production of ACTH by controlling the cleavage of POMC into its derivatives

(Adrenocorticotropic Hormone, n.d.). The highest levels of CRH was found in both rats and

human in the paraventricular nucleus and the arcuate nucleus of the hypothalamus, which is its

origin (Carr, 2013). AVP is also produced in the PVN and the two peptides, CRH and AVP, are

released from the median eminence into the hypophyseal portal system (Harno, 2016). Argenine

vasopressin itself places a weak stimulation on ACTH release, but with CRH it is a powerful

synergist in playing a role in the stress-induced release (Harno, 2016). During chronic stress, the
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ratio of AVP to CRH increased from 2:1 to as high as 9:1 as there is good evidence that AVP

acts by enhancing the responsiveness of corticotropes to CRH (Carr, 2013). ACTH release also

follows the administration of several neurotransmitters, including norepinephrine, epinephrine,

serotonin, and acetylcholine, and a variety of the nonapeptides (OXY, VIP, PHI, Ang-II) but

none of these have a major importance (Carr, 2013). Negative feedback systems regulate the

amount of ACTH and cortisol in the blood. Higher than normal blood levels of cortisol and other

adrenal hormone products inhibit the further release of both CRH and ACTH on the

hypothalamic and pituitary level (Adrenocorticotropic Hormone, n.d.).

Corticotropin releasing hormone is regulated not only by negative feedback but by other

hormones. These hormones include L-dopa, serotonin, GABA, opiate receptor agonists,

oxytocin. Serotonin and L-dopa both work on the paraventricular nucleus by increasing the

neuronal release of CRH, which in turn would produce more ACTH (Harno, 2016). In rats,

gamma-aminobutyric acid, GABA, has a direct inhibitory effect on CRH and corticotropes but in

humans, GABA stimulates the release of ACTH with the extrahypothalamic GABA inhibiting

ACTH secretion (Carr, 2013). Opiate receptor agonists effects ACTH by inhibiting the release of

CRH at the hypothalamus (Harno, 2016). Oxytocin can have different effects depending on the

species. In humans, oxytocin inhibits secretion of ACTH stimulated by CRH but in rats, oxytocin

binds to AVP receptors and stimulates the release of ACTH (Harno, 2016).

Corticotropin-releasing hormone starts the process of producing ACTH in corticotropes

of the pars distalis. ACTH is a category III tropic hormone, which means it is a POMC

derivative. POMC is produced primarily in the neurons of the hypothalamic arcuate nucleus

where the peptides are central to the regulation of energy and food intake, but POMC is

expressed in other portions of the body (Harno, 2016). POMC is located in the anterior pituitary
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and the cleavage is begun in the Golgi apparatus of corticotropes (Harno, 2016). Figure 2

demonstrates the POMC processing and release of ACTH. Prohormone convertase 1, PC1,

cleaves POMC in the pars distalis at basic residues and results in ACTH, -lipotropin and -

endorphin (Carr, 2013). PC2 cleaves further in the pars intermedia to result in -MSH, CLIP, -

LPH and -END (Carr, 2013). Studies involving the mouse pituitary tumor cell line suggest that

the cleavage of POMC is sequential with the cleavage starting at the C-terminus of ACTH

(Harno, 2016).

Figure 2: POMC Processing

involving the Golgi apparatus

and ACTH export (Harno,

2016)

Adrenocorticotropic hormone from the corticotrophs of the pars distalis is released into

the bloodstream and travels to its target, the adrenal glands. ACTH acts on a G-protein coupled

receptor called melanocortin-2 receptor to stimulate the release of glucocorticoids including

cortisol (Carr, 2013). A melanocortin receptor accessory protein is requires for the MC2R to be
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functionally active (Fridmanis, 2017). MC2Rs are also referred to as ACTHRs and is the most

specialized receptor of the melanocortin receptor family due to its tissue distribution profile in

which it only binds adrenocorticotropic hormone (Fridmanis, 2017). Other melanocortin

receptors can bind ACTH, and some were found by research groups in melanocytes (Fridmanis,

2017). Specifically, most ACTHRs are found on the zona fasciculata of the adrenal cortex, with

some found on the reticularis (Fridmanis, 2017). The ACTH stimulation of the zona reticularis

stimulates the adrenal androgen production of DHEA, DHEA-S, and androstenedione (Carr,

2013). In the zona fasciculata, adrenocorticotropic hormone stimulates the secretion of the

glucocorticoid, cortisol (Fridmanis, 2017). The zona fasciculata is the largest of the three zones

of the adrenal cortex and consists of polyhedral cells that are the source of glucocorticoid

production (Carr, 2013). The primary glucocorticoid produced, cortisol, deals with stress

(Adrenocorticotropic Hormone, n.d.). The cells of the zona fasciculata are arranged in narrow

columns, or fascicles, that surround blood sinusoids allowing the cells to be continuously bathed

with blood (Carr, 2013). The thickness of the zona fasciculata is dependent on the circulating

levels of ACTH with atrophy occurring with prolonged glucocorticoid levels or hypophysectomy

and hypertrophy occurring with prolonged ACTH levels (Carr, 2017). The third portion of the

adrenal cortex, the zona glomerulosa produces aldosterone. Although adrenocorticotropic

hormone is not necessary for the production and release of aldosterone, responsiveness of the

glomerulosa cells is enhanced with ACTH treatment and reduced in hypophysectomized animals

(Carr, 2013). This means that although ACTH does not have a clear direct role there are still

aspects of the hormone that influence the production of aldosterone that may have a role in some

aspects of the pathophysiology of conditions involving adrenocorticotropic hormone.

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The cortisol produced by stimulation of the hypothalamic-pituitary-adrenal axis helps to

deal with different types of stress. Stress includes exercise, acute illness, surgical stress,

hemorrhage and hypoglycemia but also chronic stress (Harno, 2016). The chronic stress presents

constant activation of the axis and can be linked to failures in regulation of normal feedback

(Fridmanis, 2017). Cortisol has a variety of functions but deals primarily with the bodys use of

glucose and lipids for energy and helps to reduce inflammation.

Malfunctions in the body can occur if there is too much adrenocorticotropic hormone

produced or too little. Conditions characterized with hyposecretions include that of Addisons

disease, and congenital adrenal hyperplasia (Adrenocorticotropic Hormone, n.d.). Conditions

characterized with a hypersecretion of ACTH include that of Cushings disease, Cushings

syndrome, and a rare condition, Nelson syndrome (Adrenocorticotropic Hormone, n.d.).

Addisons disease is a rare early onset autosomal disorder resulting in low cortisol levels,

but excess ACTH levels (Fridmanis, 2017). The low cortisol levels are due to failure of the

adrenal glands and the production of cortisol as well as aldosterone slows (Addisons Disease,

n.d.). The result of the low cortisol leads to hypersecretion of ACTH to attempt to raise the

cortisol levels (Addisons Disease, n.d.). The hypoplasia of the adrenals and the resulting

excess of ACTH lead to hypoglycemia and excessive skin pigmentation as well as other side

effects and symptoms including fatigue and low blood pressure (Fridmanis, 2017). The excessive

skin pigmentation is due to ACTHs cleaving into -MSH, but also ACTH independently

binding to melanocortin receptors in the skin (Harno, 2016). The lower levels or absence of

aldosterone causes the weakness, low blood pressure and water loss and can cause a salt craving

to try and restore the balance (Carr, 2013). The most common cause if the autoimmune attack

that results in bilateral atrophy of the adrenal glands (Carr, 2017). Another cause is a mutation in
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the melanocortin-receptor accessory protein, resulting in type 2, and type 3 is the unknown

causes (Fridmanis, 2017). Addisons disease has the ability to occur in all age groups and effects

both sexes (Addisons Disease, n.d.) Patients with Addisons disease exhibited a positive

response to glucocorticoid treatment (Fridmanis, 2017). The glucocorticoid treatment comes in

options including that or oral corticosteroids and also corticosteroid injections (Addisons

disease, n.d.). Adding cortisol levels to the body will result in

Congenital adrenal hyperplasia is a group of genetic defects and disorders in the genes

that code for the enzymes used in corticosteroid synthesis (Carr, 2013). The corticosteroid

synthesis can be slowed resulting in a decrease of cortisol levels and a resulting increase in

ACTH levels. The higher ACTH levels can result in hypertrophy and hyperplasia of the adrenal

cortex (Carr, 2013). CAH can also affect the production of other steroid hormones including that

of aldosterone and androgens, but in most cases results in low cortisol and excess androgens

(Congenital adrenal hyperplasia, n.d.). There are two levels, the classic and nonclassic with the

classic being more severe and detected in infancy and the nonclassic being detected in childhood

to early adulthood (Congenital adrenal hyperplasia, n.d.). The common, or classic form is

caused by the absence of 21-hydroxylase which blocks the synthesis of cortisol, corticosterone

and aldosterone leading in salt loss as a major characteristic (Carr, 2013). Nonclassic CAH, or

cryptic CAH is described as excessive androgen production later in life caused by C21-

hydroxylase deficiency but may only involve cortisol levels (Carr, 2013).

HYPERSECRETION
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Cushings disease and Cushings syndrome are both very similar with the main difference

on the source of the excess ACTH. Cushings disease was first described in 1932 by Cushing, is

the result of a pituitary adenoma not sensitive to feedback control that in turn hypersecretes

ACTH (Carr, 2013). Excess ACTH caused the adrenal gland to secrete cortisol (Cushings

Syndrome and Disease, n.d.) Cortisol levels are elevated and effects metabolism as well as

other bodily functions (Carr, 2013). Hyperpigmentation may also occur due to excess ACTH

levels (Carr, 2013). Cushings disease is usually the major cause of Cushings syndrome with the

syndrome referring to an excess of cortisol in the body (Cushings Syndrome and Disease,

n.d.). Cushings syndrome may be caused by non-pituitary adenomas or adrenal adenomas (Carr,

2013). Normal levels of ACTH may be present, but adrenal adenomas may act in the

overproduction of cortisol (Harno, 2016). Hyperpigmentation only occurs in the pituitary-

dependent form (Carr, 2013). Cushings syndrome may also be caused as the result of

glucocorticoid therapy and can have various symptoms including high blood pressure, high

blood glucose levels, fatigue, weight gain and diabetes (Kahn, 2016). Treatment of Cushings

syndrome depends on the cause but medication used to decrease cortisol production or ACTH

production can be prescribed as well as medications that block the effect of cortisol (Kahn,

2016). Tumors may be removed or if in the case that the syndrome was caused by

glucocorticoids, a change in medicine or dosage may help (Kahn, 2016). Both of these are more

common in females as compared to men.

Nelson Syndrome is the signs and symptoms from an ACTH-secreting pituitary adenoma

after a bilateral adrenalectomy (Wilson, 2015). Most cases result from the adrenalectomy in

patients who have Cushings disease (Wilson, 2015). After the cortisol levels have been leveled,

an increase in CRH occurs resulting in growth of the original tumor (Wilson, 2015). Nelson
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syndrome; however, is a rare disorder and is often not diagnosed even though it may be seen in

8-44% of patients of Cushings disease that underwent bilateral adrenalectomy (Wilson, 2015).

Adrenocorticotropic hormone has been experimented with many times in order to come

to a conclusion about what the hormone does. Below are examples in both human and non-

human vertebrate models dealing with the mechanism or effects, and pathophysiology of

adrenocorticotropic hormone. The research papers analyzed are outlined in a fashion to explicitly

explain and show the author, title, source, hypothesis, experimental design, results and

conclusions. A summary of each research paper will follow each outline that includes how the

paper is related to the assigned topic.

Mechanism/Effects of ACTH with HPA-axis suppression -HUMAN

o Author

Riikka Karlsson, Jssns Ksllio, Kerttu Irjala, Satu Ekblad, Jorma Toppari,

Pentti Kero

o Title

Adrenocorticotropic and Corticotropin-Releasing Hormone Tests in

Preterm Infants

o Link

https://academic.oup.com/jcem/article-

lookup/doi/10.1210/jcem.85.12.7032

DOI - https://doi.org/10.1210/jcem.85.12.7032

o Hypothesis

CRH stimulation tests work more reliability to reveal HPA-axis

suppression in infants.
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o Experimental design

Eighteen neonatal intensive care unit patients receiving dexamethasone

treatment were included in the study with the median gestational age of 30

weeks and 3 days. The infants were receiving dexamethasone treatment to

prevent and treat chronic lung disease. In the study, the infants HPA-axis

functions were tested twice. Before each test was taken, blood samples

were taken. The first day they were tested using the low-dose ACTH test,

which consisted of an iv bolus of 1 g/1.73 m2 synthetic ACTH,

Synacthen. Blood was taken after the test at 30 minutes to measure the

serum cortisol concentration. The second days test consisted of the CRH

test in which the subjects were given an iv bolus of 1 g/kg synthetic

human CRH, Corticorelin. Blood was taken after the CRH test at 15 and

60 minutes to measure the plasma ACTH (0 and 15 minutes) and serum

cortisol levels (0 and 60 minutes). All tests were performed in the morning

and none of the infants received dexamethasone during the study days.

Some infants (15 infants) were receiving replacement therapy for cortisol

suppression during the study, but it was not administered for at least 12

hours preceding the two tests. The serum cortisol concentration was

measured on Immuno 1 analyzer with heterogeneous competitive enzyme

immunoassay. The plasma ACTH concentration was measured by

immunoradiometric assay using a commercial kit.

o Results
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Both tests had similar basal cortisol concentrations in all of the patients.

The CRH test yielded 2 of the 18 patients with a proper ACTH response at

15 minutes while the remaining 16 infants had a weak ACTH response.

The ACTH secretion response was considered statistically significant

when using a P-value of 0.05. One of the 18 patients with the CRH test

had sufficient cortisol at 60 minutes. The ACTH test yielded five patients

exhibiting cortisol concentrations over the limit if 360 nmol/L. Two of the

patients that had a cortisol level over that of the limit also had an ACTH

concentration over 9 pmol/L during the CRH test. Comparatively, the

ARCH test resulted in a higher frequency and concentration of serum

cortisol levels than in CRH tests. Only one patient had normal HPA-axis

function according to both the CRH and ACTH tests (number 12). Four

patients had normal functioning HPA-axis with the ACTH test alone along

the parameters of the lower limit of cortisol, 360 nmol/L. If 560 nmol/L

was considered to be the correct lower limit of proper cortisol secretion,

only one patient would have passed the ACTH test.

o Conclusions

The conclusion of this experiment is that the ACTH tests used cannot

reliably detect the suppression after dexamethasone therapy. If stress

factors are present after a glucocorticoid treatment, the evaluation of

cortisol secretion with CRH test should be considered.

o Summary
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This experiment measured the accuracy of certain tests including the CRH

and ACTH tests to determine cortisol levels after speculation that the

ACTH tests were not accurate. Preterm infants usually experience

suppression of endogenous cortisol secretion. This means that their

hypothalamic-pituitary-adrenal axis is not working properly. Postnatal

dexamethasone therapy is usually the cause for the suppression. When the

studies were done, it tried to examine how well the infants HPA axis was

functioning while comparing the accuracy of different tests. Although the

CRH test was shown to be more accurate, the experiment showed the

effects of hormones, specifically ACTH and CRH, through the axis. It

showed that corticotropin releasing hormone results in the production of

ACTH and then cortisol. It showed through the CRH tests results, that

comparatively, it seems that lower levels in pmol/L of plasma ACTH

concentration levels yields extremely higher serum cortisol concentration,

nmol/L. This is evident in the comparison of graphs A and B when the

conversion factor that 1000 pmols is the same as 1 nmol.

Mechanism/Effects of ACTH with cows NON-HUMAN VEREBRATE

o Author

Marcela del Rosario Gonzlez-de-la-Vara, Ricardo Arturo Valdez,

Vicente Lemus-Ramirez, Juan Carlos Vzquez-Chagoyn, Alejandro

Villa-Godoy, and Marta C. Romano

o Title
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Effects of adrenocorticotropic hormone challenge and age on hair cortisol

concentrations in dairy cattle

o Link

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3122973/

o Hypothesis

Cortisol accumulates in cow hair after ACTH challenges and there is a

relationship between cortisol levels and cow hair color in Holstein cows.

o Experimental design

The animals, cows, in the study were maintained in irrigated alfalfa

pastures with a constant diet at La Santillana Ranch. The cows were

trained a month prior of the study with the experimental procedures in

order to reduce stress from handling and management. Twelve Holstein

cows were used to collect hair samples of different color. Two samples,

one white and one black, of each cow were collected on the same day and

were analyzed for cortisol content by RIA. To determine the effect on age

on hair cortisol, hair from different aged animals were compared. Six

samples were taken from 2-year-old cows and six samples were taken

from 15-day-old heifers and were analyzed for cortisol by RIA.

Fifteen Holstein heifers between 17 and 22 months and from 2 to 6

months pregnant were used. The heifers were separated into three groups

of five with one cow of each age and month of pregnancy. Hair samples

were taken from one group, 2.5 mL saline solution was injected to another

group three times and three injections of ACTH, 0.15 UI per kg of the
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cows bodyweight, were administered. Blood samples were collected

before, 60 minutes after and 90 minutes after the injection of ACTH. The

blood was centrifuged and the serum was analyzed for cortisol content

using RIA.

The black hair was collected on day 0, 14, 28 and 42 by an electric hair

clipper and were stored for 30 days until processed for cortisol content.

The hair was processed by being washed and then cut into about 2 mm

fragments. Three hundred milligrams of the hair were shaken for two

hours with methanol and 4 hours in a multitube vortexer. The supernatant

was decanted and at rest for 2 hours for sedimentation until it was

evaporated to dryness under a high purity nitrogen steam.

Cortisol concentrations in bovine serum were measured without extraction

and the cortisol in the hair was quantified in reconstituted hair extracts and

both were measured using a commercial solid phase kit. In the kit, the

cortisol specific monoclonal antibodies cross react with respect to other

endogen steroids is less than one percent. For each measurement, a

standard curve was made and the measurements were done using a gamma

ray counter.

Statistically, a 95% confidence interval was used and the analyses were

done using a computer software.

o Results

Serum cortisol concentration evaluated at 60 and 90 minutes and 0, 7 and

14 days after ACTH injection were significantly higher than that of the
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saline solution, with no differences in cortisol levels at the beginning of

the experiment.

Regarding the hair cortisol, no significant difference was found among

croups in cortisol concentration obtained on days 0 and 44. In the ACTH

group, hair cortisol levels were significantly higher in samples on days 14

and 28 compared to days 0 and 44.

When the animal age and hair color were taken into consideration the

cortisol, the was a significant difference. Cortisol concentrations were

higher in the 15 day old heifers as compared to those from two-year-old

cows. The values were 114.5 pg/mg of hair with a 14.43 variation and

12.15 pg/mg of hair with a 1.85 variation respectively. The cortisol levels

in the hair of 2-year-old cows were greater on that of the white hair as

compared to black with the values (23.84 +/- 2.51 pg/mg of hair and 14.31

+/- 1.07 pg/mg of hair respectively.

Other findings involved the finding that hair cortisol is stable for at least

11 months. It was also found that hair is a good source to identify

physiological events in animals because it it less invasive than other

procedures.

o Conclusion

In conclusion, it was found that cortisol concentration can be measured in

bovine hair by RIA. The technique allowed the analysis of other questions

and showed that white hair accumulates larger amounts of cortisol

compared to black hair. It was also found that calves have greater cortisol
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23

levels then cow hair and that the hormone accumulation in bovine hair was

influenced by 3 ACTH challenges. Overall, the results showed that cow

hair accumulates cortisol in response to ACTH and could be used as an

indicator for things that activate the HPA axis.

o Summary

This study provided a lot of information and much of it was useful in

obtaining a better understanding in both cortisol and adrenocorticotropic

hormone. It showed the varying cortisol levels with respect to age and hair

color. This study could help to start to explain some decline in endocrine

function with age. In this study it could possibly show the decline in

adrenal function. This can be seen by the lower levels of cortisol in older

cows as compared to younger cows. This experiment also showed how the

hypophyseal-pituitary-adrenal axis functions as the injected ACTH lead to

increased levels of cortisol indicating a normal response in the zona

fasciculata of the adrenal cortex. Overall, this study showed not only

information regarding cortisol information, but it showed the mechanism

and effects that ACTH has on the adrenals in the HPA axis.

Pathophysiology of ACTH, Hypoadrenocorticism ANIMALS

o Author

Y Engelbrecht, T Herselman, A Louw and P Swart

o Title
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24

Investigation of the primary cause of hypoadrenocorticism in South

African angora goats (Capra aegagrus): a comparison with Boer goats

(Capra hircus) and Merino sheep (Ovis aries).

o Link

https://www.animalsciencepublications.org/publications/jas/articles/78/2/3

71

o Hypothesis

There is a site of reduced adrenal function in Angora goats that results in a

decrease in cortisol production which will be tested using intravenous

treatments of insulin, CRF and ACTH.

o Experimental Design

Eight Angora goat does were used for the insulin test, eight Boer goat does

were used for the CRH stimulation test and seven Merino sheep ewes were

used for the ACTH stimulation test. The animals were kept at

Grootfontein Agricultural Institute with a consistent feeding process. All

the animals were 6 months old. For the primary cell cultures, the materials

needed were collected from donor animals between 2 and 6 years old and

from the Merino sheep or local farmers Angora and Boer does.

In the insulin tests, biosynthetic human insulin was diluted to 1 IU/mL in

1% NaCl solution and was given to the animal as an injection. Blood

samples were collected at 0 minutes and again at 15, 30, 60 and 90

minutes and the plasma was harvested via centrifugation. A day later, the

sheep synthetic CRF was prepared by diluting to 10 g/mL in 1% NaCl

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25

solution. The solution was injected with the blood procedure being the

same as that of the insulin procedure. On the third day, the ACTH solution

was prepared by diluting human synthetic ACTH to 100 g/mL in 1%

NaCl solution. The solution was also injected with the same blood

procedure as the other days. All plasma samples were stored at -20C until

tested.

The Angora goat, Boer goat and Merino sheep adrenals were collected

immediately after decapitation and placed on ice in a balanced salt

solution. The adrenal was sliced into thin slices with the glomerulosa and

the outer fasciculata discarded. The rest of the fasciculata and the zona

reticularis was finely chopped and and digested by incubation with

collagenase dissolved in EBSS. The incubation was for 150 minutes and

was shaken vigorously in 30 minute intervals. The dispersed cells were

cells were harvested and separated from undigested tissue by filtration and

centrifugation for 20 minutes. The pelleted cells were resuspended in

EBSS and filtered via nylon again and the process again using Sephadex.

The resulting cell suspension was centrifuged for 30 minutes and

resuspended in Hams F10 containing 10% fetal calf serum and 1 mL each

of penicillin, streptomycin and amphotericin B. Six grams of adrenal

glands yielded 72 x 106 purified cells.

The adrenal cells were incubated with pregnenolone alone and also with

pregnenolone and adrenocorticotropic hormone, cholera toxin or forskolin.

A control experiment was used in which the growth medium was used but
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26

with no pregnenolone. The cells were incubated for 72 hours and the

medium was slowly removed.

Blood cortisol concentrations were determined by a commercial RIA kit.

The cortisol was extracted from the plasma prior to the examination. The

production of cortisol by the pituitary adrenal cell structures were

determined using an RIA kit.

o Results

The intravenous injection of insulin in the Angora goats, Boer goats and

Merino sheep resulted in a decreased blood glucose levels after 30 minutes

but after 90 minutes, the blood glucose concentration levels were normal.

The artificially induced stress resulted in cortisol production with the max

presented in the Merino sheep and in the Boer goat after 60 minutes with

values both around 20 ng/mL. The Angoras maximum cortisol was only

about 10 ng/mL. When the injection of sheep CRF, the Merino increased

from an initial 15.7 to 34.6 ng/mL after 30 minutes. Both goat species did

not respond as well, but their cortisol levels slowly increased from 12 and

13 to 22 and 26 ng/mL for the Angora and Boer respectively. With the

ACTH injection, all species showed an increase in a similar fashion. The

Merinos cortisol was the highest followed by the Boer and Angora goats.

In all the species, the ACTH injection had the most profound effect on

cortisol release with no difference in the effect of insulin and CRF. With

respect to the cell cultures, the production of cortisol was compared. The

maximum endogenous cortisol production for Angora and Boer goats and
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27

Merino sheep were 1.37, 1.51 and .827 mol of cortisol respectively.

When pregnelone was added, each value of cortisol increased after 72

hours with the Boer goat 6.97 mol, Angora 3.05 mol and Merino was

2.43 mol of cortisol with the Boer goats being significantly higher. In the

Angora goat, there was no difference in the production of cortisol with or

without ACTH stimulation. After 24 hours, the ACTH stimulated Baer

goat cells produced significantly more cortisol than the cells that went

without stimulation, likewise is seen in the Merino sheep adrenal cells.

Cholers toxin increased cortisol production more than ACTH with no

difference in the stimulating effects of ACTH and forskolin in Angora

goats. There was no difference in the effect of the stimulants on cortisol

production for Boer goat cells. Cortisol production was increased in both

forskolin and cholera toxin to a greater extent than ACTH in the Merino

sheep cells.

o Conclusion

Many of the stress-related problem that the Angora goas are linked to are

result of the inability to maintain blood glucose levels under stress. The

insulin was used to stimulate stress, and was successful in that aspect. The

Angora goats did not have an increased cortisol level after insulin

injection. The lack of response supported the view of hypoadrenocorticism

in high fiber producing Angora goats. Overall the study confirms the

observed reduced adrenal function as the cause of hypoadrenocorticism in

South African Angora goats on a molecular basis. The cAMP signaling

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28

mechanism seems to be the problem that is required for enhanced

glucocorticoid production under stress.

o Summary

Hypoadrenocorticism is the underproduction of cortisol from the adrenal

gland. This experiment tested the idea that the reason for the decline in

Angora goats was due to their lack of cortisol produced in response to

stress. This experiment showed that there is a problem in the goats

adrenal gland in processing excess ACTH which could be the result of

stress. Stress normally increases cortisol levels, so the lack of increase

may be related to a malfunction or lack of stimulation on the ACTH

receptor in the zona fasciculata in the adrenal gland.

Pathophysiology of ACTH, Addisons disease HUMANS

o Author

Amina Dawoodji, Ji-Li Chen, Dawn Shepherd, Frida Dalin, Andrea

Tarlton, Mohammad Alimohammadi, Marissa Penna-Martinez, Gesine

Meyer, Anna L Mitchell, Earn H Gan, Eirik Bratland, Sophie Bensing,

Eystein Husebye, Simon H. Pearce, Klaus Badenhoop, Olle Kmpe, and

Vincenzo Cerundolo

o Title

High frequency of cytolytic 21-Hydroxylase specific CD8+ T cells in

autoimmune Addisons disease patients

o Link

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4821366/
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29

o Hypothesis

Epitopes on 21-OH are targeted by T cells in Addisons disease patients

and these cells are functionally capable of destroying the adrenal cortex.

o Experimental Design

Blood samples were taken from 21-OH-Ab-positive Addisons Disease

patients. DNA was extracted form patient PBMC for haplotyping.

Overlapping 18aa peptides were synthesized that span the whole 21-OH

sequence and were dissolved in DMSO. Patient cells were stimulated with

21-OH peptide pool in RH-10 and were pulsed for 1 hour before the

volume was increased to 2 mL. On day 3, cells were fed with RH-10 and

continued to be fed and split as necessary with IL-2 containing medium

until day 13 when they were prepared for intracellular staining assay. The

T cells underwent FASC staining. Samples were analyzed using Flowjo

and were then separated into Live CD3+ CD4+ or CD8+ cells and analyzed

for their cytokine positivity. Cells were added to a washed plate and was

incubated for 18 hours, washed again, added a biotin detection antibody

diluted in PBS and incubated again for 2 hours. Further incubation was

done and the plates were washed with cold water and dried completely

before counting spots using an AID ELISPOT. Monomers were

tetramerised using Streptavidin-APC before being used to stain T cells.

Granzyme B secretion was measured by ELISA. RNA was extracted from

21-OH expressing NCI H295 cells using an Qiagen RNAeasy kit and

DNA was synthesized using the RETROSCRIPT kit. The 21-OH fragment
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30

was amplified from prepared cDNA. About 150,000 21-OH Green

Fluorescence Protein transduced cells were placed with 150,000 un

transduced B cells that had been labeled in flat bottom well okated and co-

cultured with 21-OH specific T cellclones overnight at different

effector:target ratios. Cells were washed and stained with Invitrogen

Live/Dead dye to determine killing of target cells. The target cell survival

was calculated as the percentage of GFP positive cells compared to the

CMTMR positive cells and the target cell lysis was calculated.

o Results

Most of the 20 Addisons disease patients had high numbers of 21-OH

specific CD8+ and CD4+ T cells as compares to the baseline values found

in healthy controls. The magnitude of the 21-OH specific T cell response

decreased over time, but it was still able to detect responses in the majority

of samples from 1-2 years since their diagnosis. The majority of patients

revealed CD8+ T cells capable of recognizing 21-OH337-354 and/or 21-

OH428-445 and in contrast, a number of CD4+ T cells recognized 21-OH207-

224. The patients showed responses in the ex-vivo assay that were specific

peptides demonstrating that the T cell responses were not being biased to

particular peptides during bulk culturing with the pool of peptides. T cells

were able to lyase HLA A2+ target cells transduced with a lentiviral

vector, which confirmed their cytotoxic capacity.

o Conclusion
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Immunodominant regions of 21-Hydroxylase were identified and it was

exhibited that 21-OH specific T cells recognize endogenous 21-OH protein

and may be linked directly to the progression of Addisons disease. There

were unexpected outcomes in which the years of 21-OH T cell responses

in destroyed adrenal glands without endogenous steroid production

indicated the presence of remaining antigen stimulation.

o Summary

Overall, this study was a good example of explaining Addisons disease

and the destruction that causes it. It also gave a greater understanding of

how the adrenal cortex works and the possibility why the degeneration

occurs in Addisons disease.

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32

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