diagnostics

Review
Adrenal Failure: An Evidence-Based Diagnostic Approach
Salomi Shaikh 1,† , Lakshmi Nagendra 2,† , Shehla Shaikh 3 and Joseph M. Pappachan 4,5,6, *

1 KGN Diabetes and Endocrine Centre, Mumbai 400001, India

2 Department of Endocrinology, JSS Medical College, JSS Academy of Higher Education and Research Center,
Mysore 570015, India
3 Department of Endocrinology, Saifee Hospital, Mumbai 400004, India
4 Department of Endocrinology & Metabolism, Lancashire Teaching Hospitals NHS Trust, Preston PR2 9HT, UK
5 Faculty of Science, Manchester Metropolitan University, Manchester M15 6BH, UK
6 Faculty of Biology, Medicine and Health, The University of Manchester, Manchester M13 9PL, UK
* Correspondence: [email protected]; Tel./Fax: +44-177-252-2092
† These authors contributed equally to this work.

Abstract: The diagnosis of adrenal insufficiency (AI) requires a high index of suspicion, detailed
clinical assessment including detailed drug history, and appropriate laboratory evaluation. The
clinical characteristics of adrenal insufficiency vary according to the cause, and the presentation may
be myriad, e.g. insidious onset to a catastrophic adrenal crisis presenting with circulatory shock
and coma. Secondary adrenal insufficiency (SAI) often presents with only glucocorticoid deficiency
because aldosterone production, which is controlled by the renin angiotensin system, is usually
intact, and rarely presents with an adrenal crisis. Measurements of the basal serum cortisol at 8 am
(<140 nmol/L or 5 mcg/dL) coupled with adrenocorticotrophin (ACTH) remain the initial tests
of choice. The cosyntropin stimulation (short synacthen) test is used for the confirmation of the
diagnosis. Newer highly specific cortisol assays have reduced the cut-off points for cortisol in the
diagnosis of AI. The salivary cortisol test is increasingly being used in conditions associated with
abnormal cortisol binding globulin (CBG) levels such as pregnancy. Children and infants require
lower doses of cosyntropin for testing. 21-hydoxylase antibodies are routinely evaluated to rule
out autoimmunity, the absence of which would require secondary causes of adrenal insufficiency
to be ruled out. Testing the hypothalamic–pituitary–adrenal (HPA) axis, imaging, and ruling out
systemic causes are necessary for the diagnosis of AI. Cancer treatment with immune checkpoint
inhibitors (ICI) is an emerging cause of both primary AI and SAI and requires close follow up. Several
Citation: Shaikh, S.; Nagendra, L.;
Shaikh, S.; Pappachan, J.M. Adrenal antibodies are being implicated, but more clarity is required. We update the diagnostic evaluation of
Failure: An Evidence-Based AI in this evidence-based review.
Diagnostic Approach. Diagnostics
2023, 13, 1812. https://doi.org/ Keywords: adrenal failure; cortisol; adrenocorticotrophin (ACTH); primary adrenal insufficiency
10.3390/diagnostics13101812 (PAI); secondary adrenal insufficiency (SAI)

Academic Editor: Enrico Carmina

Received: 3 April 2023

Revised: 14 May 2023 1. Introduction
Accepted: 18 May 2023
Adrenal insufficiency (AI) is an uncommon but serious hormonal disorder which
Published: 21 May 2023
results from the complete or partial cessation of adrenal steroidogenesis to maintain normal
health and well-being of the affected individual. AI occurs as a consequence of several
diseases or insults to the adrenal glands and can present with very subtle symptomatology
Copyright: © 2023 by the authors.
to catastrophic adrenal crisis that can even endanger life [1,2]. The signs and symptoms
Licensee MDPI, Basel, Switzerland. of AI vary widely and can mimic many other systemic disorders, delaying the clinical
This article is an open access article suspicion and diagnostic evaluation. Adrenal steroid synthesis can be affected by disorders
distributed under the terms and involving the adrenal glands (primary AI), the diseases of the pituitary glands (secondary
conditions of the Creative Commons AI), or the hypothalamus (tertiary AI) with the reduced production of adrenocorticotrophin
Attribution (CC BY) license (https:// (ACTH) that stimulates adrenal steroidogenesis.
creativecommons.org/licenses/by/ Regardless of the cause of AI, a prompt and timely clinical diagnosis is crucial for the
4.0/). optimal management of patients as the delay can lead to adrenal crisis during periods of

Diagnostics 2023, 13, 1812. https://doi.org/10.3390/diagnostics13101812 https://www.mdpi.com/journal/diagnostics

Diagnostics 2023, 13, 1812 2 of 12

stress, a condition associated with high morbidity and mortality [3–6]. Moreover, though
classical cases of primary AI may not pose much diagnostic challenges to physicians and
laboratory scientists, some patients with AI can present with atypical symptoms and signs
and cause a dilemma in clinical and laboratory evaluation. Therefore, we attempt to
appraise the latest evidence on the diagnostic evaluation of the disease to enable physicians
to rationally approach any case of AI in this article.

2. Clinical Characteristics of Different Forms of AI

2.1. Primary Adrenal Insufficiency (PAI)
The clinical manifestations of PAI are influenced by the rate and degree of decline
in the adrenal function. Clinical presentation may vary widely from vague, nonspecific
symptoms of tiredness and fatigue to life-threating adrenal crisis where the patient may
become unconscious with circulatory shock and hypothermia [4,7]. Weight loss, anorexia,
nausea, vomiting, lethargy, salt craving, fatigue, muscle cramps, and postural hypotension
are typically noticeable in primary adrenal failure. About 90% of individuals with chronic
PAI have some level of skin hyperpigmentation, which is especially noticeable on the skin
creases and scars, the extensor surface of the elbow, the knuckles, the lips, and the buccal
mucosa. However, it should be emphasized that individuals with an acute onset of PAI, due
to the destruction of the adrenal glands from infarction or hemorrhage resulting from acute
vascular insults, would not have recognizable cutaneous hyperpigmentation [8]. Other
characteristics of PAI include spontaneous episodes of hypoglycemia or an unexplained
decrease in the insulin dose requirement in a person with diabetes mellitus, and in women,
axillary and pubic hair loss owing to insufficient adrenal androgen production [9].
PAI may be inherited or acquired. The most common inherited form of PAI is con-
genital adrenal hyperplasia, while autoimmunity remains the primary cause of acquired
PAI in the western world. However, various infections (bacterial, fungal, or mycobacte-
rial) causing the destruction of the adrenal glands are the most common causes of PAI
in the developing world [10,11]. Although PAI caused by autoimmune adrenalitis may
occasionally occur in isolation without other endocrine dysfunction, more than 50% of
autoimmune adrenalitis cases are associated with autoimmune polyglandular syndromes
(APS) [12]. Mycobacterium tuberculosis (MTB) is the most common infection implicated in
the development of PAI. Furthermore, infections with Neisseria meningitides, Pseudomonas
aeruginosa, Haemophilus influenzae, Pasteurella multocida, Staphylococcus aureus, and Strepto-
cocci group A, when associated with septicemia, may result in an acute presentation of
AI with bilateral adrenal infarction (known as Waterhouse–Friderichsen syndrome) often
brought on by disseminated intravascular coagulation. Fever, nausea, myalgia, arthralgia,
and an erythematous rash are the first symptoms, which proceed to cutaneous ecchymosis
and purpura fulminans in the acute stage [13]. Other rare causes that can lead to the
development of PAI include adrenal hemorrhage, metastatic cancer, infiltrative disorders,
and certain drugs such as ketoconazole, fluconazole, etomidate, and mitotane. Drugs such
as phenytoin and rifampicin increase the metabolism of cortisol and can precipitate adrenal
insufficiency in susceptible individuals [14]. Patients with PAI usually show a pronounced
impairment of aldosterone secretion from the total absence of adrenal steroidogenesis,
leading to hyponatremia, hyperkalemia, and hypotension [15], as this mineralocorticoid
hormone normally maintains the water electrolyte balance in the human body.
Adrenal crisis is an acute, often life-threatening condition that is more commonly
associated with PAI when compared to cases of central AI [4]. The incidence of adrenal crisis
is reported to be about 8% in patients with PAI [16]. The usual presentation of patients with
adrenal crisis is dramatically diminished well-being, hypotension, nausea and vomiting,
and fever that responds effectively to parenteral hydrocortisone treatment. Adrenal crisis
is most often precipitated by infections. Other factors include hyperthyroidism, physical
stress from surgery or trauma, inadvertent discontinuation of glucocorticoid medications,
intense physical exercise, and psychological stress [17]. Early diagnosis, rehydration, and
Diagnostics 2023, 13, 1812 3 of 12

steroid replacement form the cornerstones of management, without which catastrophic

outcomes including death can result.

2.2. Secondary Adrenal Insufficiency

The most prevalent subtype of adrenal insufficiency is secondary insufficiency, often
resulting from a pituitary tumor [18]. A tumor of the hypothalamic-pituitary area is the
most common endogenous cause of secondary adrenal insufficiency (SAI), and it is typically
accompanied with panhypopituitarism due to pressure effects on the pituitary or the effects
of surgery or radiation treatment for tumor removal. Less frequently, nontumoral diseases
such as infiltrative lesions, hypophysitis, infectious processes, vascular changes, traumatic
brain damage, empty sella syndrome, or genetic problems can also cause SAI [19]. Immune
checkpoint inhibitor-induced SAI is increasingly being described in the recent literature,
as these drugs are often effectively used for the treatment of various forms of cancers [3].
Clinical signs of SAI, in contrast to PAI, are only the result of glucocorticoid depletion from
ATCH deficiency, because the production of aldosterone is often unaffected. Additionally,
SAI seldom presents with an acute adrenal crisis, but an acute presentation may be brought
on by stress from an injury, illness, or surgery. Symptoms and signs secondary to the
deficiency of other anterior pituitary hormones as well as neurological signs can coexist,
especially when the cause of SAI is a pituitary tumor [20].

2.3. Tertiary Adrenal Insufficiency

Tertiary adrenal insufficiency (TAI) is characterized by hypothalamic anomalies or
dysfunction, which results in decreased corticotropin-releasing hormone (CRH) produc-
tion, and consequently, the reduced synthesis of ACTH from the anterior pituitary gland.
TAI, like SAI, is associated with diminished cortisol and dehydroepiandrosterone (DHEA)
production from the adrenal glands because of a lack of ACTH stimulation. As a result,
TAI is commonly referred to as central AI, or sometimes as SAI, because additional dis-
tinctive features between these two forms are sparse. TAI generally develops from the
suppression of the hypothalamic–pituitary–adrenal (HPA) axis by the administration of
supraphysiological doses of exogenous steroids over a prolonged period of time [21].

3. Laboratory Evaluation for Primary AI

3.1. 8:00 a.m. Cortisol
There should be a high index of suspicion for PAI in acutely unwell patients exhibiting
clinical and biochemical features indicative of primary adrenal insufficiency (PAI), such as
volume depletion, hypotension, hyponatremia, hyperkalemia, fever, abdominal discomfort,
hyperpigmentation, or hypoglycemia (particularly in children). Treatment should not be
delayed in patients who exhibit signs and symptoms of PAI, especially when they are
acutely unwell, as delays may be associated with the development of catastrophic adrenal
crisis. A baseline blood sample may be drawn for diagnostic testing and treatment with
glucocorticoids, and prompt rehydration must be started without delay while awaiting test
results [22].
Low morning cortisol concentrations (measured in serum or plasma before 8:00 a.m.)
confirmed by low stimulated cortisol levels have typically been used to make the diagnosis
of PAI. Though the optimal basal cortisol that can confirm or rule out adrenal insufficiency
is a matter of debate, a morning serum cortisol of <140 nmol/L (5 µg/dL) in combination
with increased ACTH levels (twice higher than the upper limit of normal laboratory values)
are usually taken as confirmation of PAI [23]. A recent study demonstrated that a basal
cortisol level of >12.83 µg/dL (354 nmol/L) usually rules out chronic PAI in an individual
without stress factors such as infections [24] (see Figure 1). If only a basal cortisol test before
9:00 a.m. is performed, values of <100 nmol/L strongly suggest PAI [25].
Diagnostics 2023, 13, 1812 4 of 13

Diagnostics 2023, 13, 1812 individual without stress factors such as infections [24] (see Figure 1). If only a basal cor-4 of 12
tisol test before 9:00 a.m. is performed, values of <100 nmol/L strongly suggest PAI [25].

Figure 1. Biochemical
Figure screening
1. Biochemical evaluation
screening for suspected
evaluation adrenal
for suspected insufficiency
adrenal (AI). ACTH:
insufficiency adreno-
(AI). ACTH: adreno-
corticotrophin; HPA axis: hypothalamic–pituitary–adrenal axis; SAI: secondary adrenal insuffi-
corticotrophin; HPA axis: hypothalamic–pituitary–adrenal axis; SAI: secondary adrenal insufficiency;
ciency; ULN: upper limit of normal.
ULN: upper limit of normal.

Cortisol is predominantly
Cortisol is predominantly transported
transportedininthe
the body
body viaviacortisol-binding
cortisol-bindingglobulin
globulin (CBG)
(CBG) and albumin; only a small fraction (~10%) of the total serum cortisol
and albumin; only a small fraction (~10%) of the total serum cortisol is unbound and is unbound
and biologically
biologically active.
active.Serum
Serumcortisol
cortisolassays
assaysmeasure
measurethe the total
total cortisol
cortisol (bound
(bound andandfreefree
forms),
forms), and their results can be misrepresentative in patients with altered serum
and their results can be misrepresentative in patients with altered serum protein concen- protein
concentrations
trations [26].[26].Salivary
Salivarycortisol
cortisolwaswas shown
shown to to correlate
correlate well
wellwith
witha aserum-free
serum-free corti-
cortisol
sol level
leveland
andwaswasreported
reported asas
a promising
a promising alternative testtest
alternative for for
the the
diagnosis of AI,
diagnosis of especially
AI, especially
among patients
among with with
patients altered CBG CBG
altered concentrations [27]. A[27].
concentrations basal
A morning salivary
basal morning cortisolcortisol
salivary of
<1.0 of
nmol/L may suggest
<1.0 nmol/L PAI, while
may suggest PAI,those
whilewith values
those withofvalues
>5.9 nmol/L
of >5.9 should
nmol/Lhave a con-
should have a
firmatory stimulation
confirmatory test [28].test [28].
stimulation

3.2. Basal
3.2. Basal ACTH ACTH Measurement
Measurement
When When the cortisol
the cortisol production
production from thefrom the adrenal
adrenal glands is glands
reducedis reduced in individuals
in individuals with
with PAI, their hypothalamic–pituitary–adrenal axis is activated by the
PAI, their hypothalamic–pituitary–adrenal axis is activated by the stimulation of the hy- stimulation of the
pothalamus and anterior pituitary from low plasma cortisol through the feedback looploop
hypothalamus and anterior pituitary from low plasma cortisol through the feedback
(Figure
(Figure 2). This
2). This results
results in aninincreased
an increased production
production ofcorticotropin-releasing
of the the corticotropin-releasing hormone
hormone
(CRH)(CRH)
and and
ACTH.ACTH. An ACTH
An ACTH levellevel
that that is more
is more thanthan
doubledouble the upper
the upper limitlimit
of theofnormal
the normal
laboratory
laboratory rangerange in presence
in the the presence of low
of low basalbasal plasma
plasma cortisol
cortisol usually
usually indicates
indicates PAI [29].
PAI [29].
One needs to ensure that patients are not on corticosteroid therapy, including topical as as
One needs to ensure that patients are not on corticosteroid therapy, including topical
wellwell as inhaled
as inhaled formsforms of drugs,
of the the drugs, before
before evaluating
evaluating for PAI.
for PAI.
3.3. Stress Testing for Proving PAI
When the basal cortisol level is indeterminate (<354 nmol/L), a confirmatory test to
prove AI is necessary. A short synacthen test (SST) or cosyntropin stimulation test are
the most widely used stress testing methods to prove or disprove PAI. After securing the
baseline plasma ACTH (if not tested earlier) and cortisol samples, 250 mcg of synacthen
(cosyntropin; synthetic ACTH) is administered intravenously/intramuscularly with the col-
lection of blood samples again at 30 and/or 60 min for estimating the plasma cortisol levels.
Diagnostics 2023, 13, 1812 5 of 12

A stimulated peek cortisol level of ≥500 nmol/L (18 µg/dL) excludes adrenal insufficiency
as per the current international guidelines [23]. However, we have to bear in mind that
when using newer highly specific cortisol assays such as liquid chromatography–tandem
Diagnostics 2023, 13, 1812 5 of 13
mass spectrometry (LC–MS/MS), the cut-off values can be lower at 412 and 485 nmol/L at
30 and 60 min (of stress testing), respectively, as shown by the newer studies [30].

Figure 2.
2. Alterations
Alterationsininhypothalamic–pituitary–adrenal
hypothalamic–pituitary–adrenal axis
axis in various
in various forms
forms of adrenal
of adrenal insuffi-
insufficiency.
ciency.
ACTH: ACTH: adrenocorticotrophin;
adrenocorticotrophin; CRH: corticotrophin-releasing
CRH: corticotrophin-releasing hormone;hormone;
DHEAS:DHEAS: dehydroe-
dehydroepiandros-
piandrosterone
terone sulphate;sulphate; Red arrows
Red arrows indicateindicate site of disease;
site of disease; Blue arrows
Blue arrows indicate
indicate high high (upward)
(upward) or
or low
low (downward) hormone
(downward) hormone levels. levels.

3.3. Stress Testing forearlier,

As mentioned Provingvariations
PAI in the CBG levels can affect the usual cortisol assays,
as these
Whentests measure
the the totallevel
basal cortisol serum cortisol level. Therefore,
is indeterminate in conditions
(<354 nmol/L), with abnormal
a confirmatory test to
CBG levels such as pregnancy, nephrotic syndrome, liver cirrhosis, and
prove AI is necessary. A short synacthen test (SST) or cosyntropin stimulation test are the estrogen therapy,
the serum
most widely cortisol values
used stress may not
testing be fully
methods to reliable
prove ortodisprove
diagnose AI After
PAI. [31]. In such situations,
securing the base-
a salivary cortisol assay can be used as a reliable alternative test.
line plasma ACTH (if not tested earlier) and cortisol samples, 250 mcg of synacthen A very recent study
(co-
demonstrated
syntropin; a sensitivity
synthetic ACTH)ofis90.5% and specificity
administered of 76.2% after SST for diagnosing
intravenously/intramuscularly with the col-AI
using aof
lection stimulated
blood samplessalivary cortisol
again at 30cut-off
and/orvalue
60 min ≥16estimating
of for nmol/L [32].the plasma cortisol lev-
els. AThe gold standard
stimulated dynamiclevel
peek cortisol test of
for≥500
assessing
nmol/L the(18
entire HPAexcludes
µg/dL) axis andadrenal
adrenalinsuffi-
failure
is an insulin tolerance test (ITT). Hypoglycemia is one of the most powerful
ciency as per the current international guidelines [23]. However, we have to bear in mind stressors for
adrenal steroid secretion. The test is performed using intravenous insulin
that when using newer highly specific cortisol assays such as liquid chromatography– (0.1–0.15 unit/kg
body weight)
tandem mass administration to induce hypoglycemia
spectrometry (LC–MS/MS), the cut-off(plasma
values glucose <2.2 mmol/L),
can be lower at 412 and which
485
results in a cortisol surge from the adrenal glands [29,33]. An ITT must
nmol/L at 30 and 60 min (of stress testing), respectively, as shown by the newer studies be considered when
the SST results are equivocal, or its diagnostic predictive value is doubtful. However, testing
[30].
can beAsdifficult
mentioned in patients
earlier, with cardiovascular
variations in the CBGdisorders
levels can(such
affectastheischemic heart disease)
usual cortisol assays,
and neurological illnesses (contraindicated in patients with epilepsy as hypoglycemia of
as these tests measure the total serum cortisol level. Therefore, in conditions with abnor-
<2.2 mmol/L may precipitate a seizure episode). The overnight metyrapone stimulation
mal CBG levels such as pregnancy, nephrotic syndrome, liver cirrhosis, and estrogen ther-
test, which was used as an alternative to ITT in the past, is not often used in current clinical
apy, the serum cortisol values may not be fully reliable to diagnose AI [31]. In such situa-
practice [29].
tions, a salivary cortisol assay can be used as a reliable alternative test. A very recent study
demonstrated a sensitivity of 90.5% and specificity of 76.2% after SST for diagnosing AI
using a stimulated salivary cortisol cut-off value of ≥16 nmol/L [32].
The gold standard dynamic test for assessing the entire HPA axis and adrenal failure
is an insulin tolerance test (ITT). Hypoglycemia is one of the most powerful stressors for
adrenal steroid secretion. The test is performed using intravenous insulin (0.1–0.15 unit/kg
body weight) administration to induce hypoglycemia (plasma glucose <2.2 mmol/L),
Diagnostics 2023, 13, 1812 6 of 12

3.4. Other Laboratory Abnormalities in PAI

It is important to investigate the underlying cause of PAI for establishing the patholog-
ical diagnosis, as that has therapeutic and prognostic implications. As autoimmunity is the
major cause of acquired PAI in the western world, 21-hydroxylase antibodies should ideally
be checked in all cases [29]. If the patient tests positive for the antibodies, the patient should
be further investigated for other autoimmune diseases such as type 1 diabetes mellitus,
thyroid disease, premature ovarian failure, celiac disease, and atrophic gastritis with B12
deficiency, as PAI may be part of APS [34]. Organ-specific autoimmune disorders affecting
other endocrine organs/body tissues may co-exist in patients with 21-hydroxylase auto-
antibodies, or such autoimmunity can develop in these individuals in later life. Therefore,
the current guideline of the Endocrine Society also recommends periodic testing for these
conditions, ideally on an annual basis, if these tests are initially negative [23].
In the absence of adrenal autoantibodies, patients should be further investigated
for structural defects in the glands such as infections, tumor infiltration, and bleed. A
computed tomographic (CT) imaging is recommended, and other investigations such
as testing for mycobacterial disease and fungal infections (in endemic areas) should be
pursued according to the clinical need. Congenital disorders of adrenal steroidogenesis
should be considered in patients younger than 20 years of age, and they should be worked
up, as shown in Table 1. Molecular and genetic testing for these cases should help with
planning definite management and prognostication.

Table 1. Various forms of adrenal insufficiency, the causes, and diagnostic evaluation. AI: adrenal
insufficiency; APS: autoimmune polyglandular syndrome; APLA: antiphospholipid antibody;
CAH: congenital adrenal hyperplasia; CHF: congestive heart failure, CMV: cytomegalovirus; CRH:
corticotrophin-releasing hormone; CT: computed tomography; MRI: magnetic resonance imaging;
VLCFA: very long chain fatty acid; * after ruling out pheochromocytoma.

Disease Entity Cause Diagnostic Evaluation

High baseline ACTH, low basal & stimulated
Primary AI
serum cortisol
• Autoimmune AI
Isolated autoimmune adrenalitis
Associated with APS-I Immune reaction against
Autoantibodies against 21-hydroxylase
Associated with APS-II 21-hydroxylase
• Infection

Tuberculosis Mycobacterium tuberculosis

CT
Antigen detection tests/blood cultures
HIV Opportunistic infections/neoplasms
Fungal Histoplasmosis
Paracoccidioidomycosis
Viral CMV
• Bilateral adrenal hemorrhage
Sepsis
CHF CT
APLA
Use of anticoagulants
Trauma
Surgery

• Adrenal metastasis

Malignancy CT/biopsy *
Diagnostics 2023, 13, 1812 7 of 12

Table 1. Cont.

Disease Entity Cause Diagnostic Evaluation

• Adrenal infiltration

Amyloidosis CT/biopsy *
Sarcoidosis
Lymphoma
• Genetic AI
X-linked adrenoleukodystrophy VLCFA
CAH 17-OHP
Adrenal hypoplasia congenita NR0B1 mutation
ACTH insensitivity syndrome MC2R mutation

• Drug induced Various adrenolytic agents Prompt medication history

Secondary AI Low basal ACTH, low stimulated serum cortisol

• Space occupying lesions Pituitary tumors MRI
• Trauma Altered blood supply MRI
• Surgery/irradiation MRI

• Infection
Tubercular hypophysitis MRI/biopsy
Pyogenic abscess

• Infiltration
Lymphocytic hypophysitis MRI/biopsy
Sarcoidosis
Wegener’s granulomatosis
Histiocytosis X

• Sheehan’s Severe blood loss/hypotension causing

MRI
necrosis of the pituitary

Ketoconazole
• Drugs Fluconazole
Etomidate
Metyrapone

Low basal ACTH, low stimulated cortisol

Tertiary AI Increased ACTH and cortisol response to CRH
stimulation test
• Space occupying lesions Hypothalamic tumors
• Trauma MRI
• Surgery
• Irradiation
• Drug induced Glucocorticoids

PAI is usually associated with a concomitant deficiency of other adrenal steroids, and
therefore, low aldosterone and DHEA levels are expected in most cases [29]. Hypoal-
dosteronism is associated with raised plasma renin activity, the measurement of which
helps in monitoring the dose titration of mineralocorticoid replacement in such patients.
Hyperkalemia and hyponatremia are manifestations of hypoaldosteronism classically seen
in patients with PAI, whereas these abnormalities are uncommon in SAI and TAI (as aldos-
terone production is not just under the control of ACTH). Eosinophilia may be a common
feature and a sensitive marker of AI [35].
Diagnostics 2023, 13, 1812 8 of 12

3.5. Special Populations with PAI

The hormone profile may be different, and biochemical confounders in hormone assays
may occur in some groups of patients such as pregnant women and children. Pregnancy
is a physiological state of hypercortisolism, and the total cortisol level may be 2–3 times
higher than normal in pregnant women due to increased levels of CBG as a consequence of
stimulation from hyperestrogenemia, especially in the third trimester [29,36]. The cut-off
levels for serum cortisol are not well-established in pregnant women, and therefore, the
diagnostic evaluation for AI can be challenging both by basal hormone measurement and
stress testing. Although the cut-off levels for pregnancy are not well-established, a salivary
cortisol assay may be utilized for the diagnostic evaluation of AI in pregnancy [29,37].
In situations where gross alterations in the serum CBG levels can occur, as in patients
with cirrhosis of liver, estrogen therapy, and nephrotic syndrome, an evaluation for AI
with total plasma cortisol levels can pose diagnostic challenges. In such situations, both
basal and stress-induced (e.g., SST) salivary cortisol measurements would provide effective
alternate testing options [38]. In children and infants, lower doses of cosyntropin must be
used for SST. In infants, cosyntropin 15 µg/kg body weight should be used, and children
<2 years should be tested with 125 µg of cosyntropin [39].

4. Diagnostic Evaluation of SAI

4.1. Biochemical Tests
As SAI is usually a consequence of pituitary disease, abnormalities of other hormones
from the gland may also co-exist in such patients. The degree of hormone abnormalities
depends on the pathological alterations in the pituitary. For example, pituitary adenomas
usually cause pressure-related damage to hormone-producing cells depending on the
tumor size and rapidity of growth of the tumor. An overnight fasting static pituitary
profile, ideally at 8:00 a.m., to estimate cortisol (+/− ACTH), gonadotropins coupled with
gonadal hormones (estrogen in females and testosterone in males), prolactin, thyrotropin
coupled with thyroxine, and insulin-like growth factor 1 (IGF-1 +/− growth hormone)
levels should be performed for a baseline evaluation [40]. The hormone deficiency or
excess state revealed by this baseline test will help further hormonal evaluation for proving
the biochemical tests. Isolated ACTH deficiency causing SAI is usually a diagnosis of
exclusion [29]. Provocative hormone tests to prove SAI can be performed using the similar
methods discussed in the previous section on PAI. The ACTH stimulation test should be
performed 6 weeks after surgery and at least 18 h after the last dose of hydrocortisone [40].
Individuals with SAI have lower z-scores for DHEAS, which has potential diagnostic value
in younger patients and patients with tumors in assessing the integrity of the HPA axis, but
more data is needed.
Pituitary insults causing SAI can also be the result of various systemic disorders such
as hemochromatosis, sarcoidosis, immunoglobulin-4 (IgG-4) related systemic disease, and
infectious diseases. Biochemical and immunological evaluations for such disorders should
be performed as per the standard recommendations (not discussed here) for arriving at an
appropriate diagnosis.

4.2. Imaging Studies

The standard diagnostic evaluation for pituitary disease following the biochemical
diagnosis of SAI is a magnetic resonance imaging (MRI) of the gland. Screening pituitary
via MRI prior to a biochemical diagnosis of SAI should be dissuaded, as the detection of
an incidentaloma is likely in up to 10–40% of such imaging studies, [41] and unnecessary
laboratory work-up and patient discomfort about the abnormal finding can be avoided.
A review of the pituitary MRI by an expert neuroradiologist can give reasonable clue to a
pathological diagnosis when a structural lesion is identified by such an imaging study in
the presence of an appropriate pre-imaging biochemical diagnosis. Functioning and non-
functioning pituitary adenomas, pituitary metastasis from primary malignancy at distal
sites, hypophysitis, infections, and granulomatous disease can all result in SAI, and addi-
Diagnostics 2023, 13, 1812 9 of 12

tional imaging studies such as CT imaging of the chest, abdomen, and pelvis, and positron
emission tomography (PET) coupled with biochemical, immunological, and microbiological
evaluation for such disorders should help in clinching the accurate diagnosis [42].

4.3. Evaluation for TAI

As TAI is usually a consequence of HPA axis suppression, a prompt clinical history
should be obtained, including the use of topical and inhaled steroids; history of recent or
current use of Indigenous medications; nutritional history including food supplements
(and their contents); chronic administration of opioids; and self-administration of over-the-
counter medications. The evaluation for various rare genetic syndromes associated with
TAI (e.g., Prader–Willi syndrome) in patients with these syndromic features is indicated
when appropriate. In addition, obtaining details about daily lifestyle including physical
activities and various physical and emotional stressors are important, as these factors may
be associated with marked alterations in the HPA axis [43].

4.4. Biochemical Work-Up

Hormonal evaluation usually involves the biochemical confirmation of AI, as described
in the previous sections. A static pituitary profile with a normal or subnormal ACTH level
with or without other pituitary hormone abnormalities may be present in patients with TAI.
A lack of ACTH and cortisol response to ITT, and an increase in these hormone outputs
following a CRH stimulation test, may point toward supra-hypophyseal disease causing
TAI [44]. As the hypothalamus also controls the posterior pituitary hormonal balance,
patients with TAI often have diabetes insipidus as a consequence.

4.5. Further Diagnostic Evaluation

Structural lesions including tumors and infiltrative lesions can often be diagnosed via
MRI of the brainstem with a pituitary protocol to investigate patients with biochemically
proven TAI. This should also aid in therapeutic decision making and prognostication [45].
Genetic studies are necessary in syndromic cases to prove the diagnosis, and for counselling
purposes [46].

5. Immune Check Point Inhibitors (ICI) and AI

Cancer treatment has been revolutionized in recent years with the advent of ICIs. As
these drugs can grossly modify the immune response in the human body, including that
in the endocrine systems, the therapeutic use of many of these agents is associated with
marked alterations in the body’s hormonal balance. Both the hyper- and hypofunction
of several endocrine glands can occur with the use of ICIs [47]. Several agents of the ICI
class have been used for cancer treatment in recent years, and many new molecules are
under evaluation in clinical trials. Ipilimumab, pembrolizumab, nivolumab, atezolizumab,
avelumab, durvalumab, and cemiplimab are some of the commonly used ICIs in current
clinical practice. Different endocrine side effects varying from mild asymptomatic thyroid
dysfunction to fatal adrenal crisis or diabetic ketoacidosis have been described with the use
of this novel class of anticancer agents, and these endocrine toxicities may occur in 25–50%
of patients [48].
Both PAI and SAI are common and can present after varying time intervals following
the initiation of ICI therapy [47–50]. Though routine screening for asymptomatic cases is
controversial, some centers screen patients for AI following every cycle of cancer treatment
with ICIs [48]. PAI usually develops as a consequence of adrenalitis, whereas SAI can
develop from hypophysitis or isolated ACTH deficiency [49]. Several autoantibodies have
been characterized in patients with ICI-induced PAI and SAI [50]. Those associated with
PAI are anti-21-hydroxylase, anti-17α-hydroxylase, anti-P450 side chain cleavage enzyme,
anti-aromatic L-amino acid decarboxylase (AADC), anti-interferon (IFN)α, and anti-IFNΩ
antibodies. Antibodies associated with SAI include anti-guanine nucleotide-binding protein
G(olf) subunit alpha (GNAL), anti-pro-opiomelanocortin (POMC), anti-TPIT (corticotroph-
Diagnostics 2023, 13, 1812 10 of 12

specific transcription factor), anti-integral membrane protein 2B (ITM2B), anti-zinc finger

CCHC-type containing 8 (ZCCHC8), and anti-pituitary-specific transcriptional factor-1
(PIT-1). However, the utility of these antibodies for routine clinical evaluation and screening
are not yet clear.
Several endocrine societies have developed guidelines for the diagnosis and manage-
ment of ICI-related endocrine dysfunction (beyond the scope of this review) and can be
utilized as per the demand of the clinical situation [51–54].

6. Areas of Uncertainty
Although several clinical practice guidelines elaborate the diagnostic approach to
various types of adrenal failure, there is no clear consensus on the cut-off levels for basal and
stimulated cortisol in special circumstances where CBG levels are altered (e.g., pregnancy,
estrogen therapy, cirrhosis, and nephrotic syndrome). There is still uncertainty about the
exact cut-off levels for the suggested alternative test, salivary cortisol (basal and stimulated)
test, in these situations, as there is only meagre evidence of its clinical utility. Similarly,
testing algorithms and the utility of various antibodies for diagnostic evaluation of patients
with ICI-induced AI is still evolving, especially because newer molecules are rapidly being
added to this group of anticancer agents.

7. Conclusions
Adrenal failure is an uncommon but life-threatening endocrinopathy with protean
manifestations. The diagnosis can often be missed without prompt clinical evaluation and
diagnostic work-up and may lead to devastating complications in the form of lethal adrenal
crisis. The clinical profile and laboratory work-up for the disease vary according to the type
of insult, namely, PAI, SAI, or TAI. Prompt assessment of the basal and stimulated cortisol
level followed by antibody testing, testing for the integrity HPA axis, various imaging
techniques, and testing for systemic insults as causes of AI are the usual approaches to di-
agnostic evaluation. Following various international clinical guidelines, when appropriate,
also helps us to plan appropriate laboratory evaluations of this uncommon but important
endocrine disorder.

Author Contributions: Conceptualization, J.M.P.; Literature search and drafting, S.S. (Salomi Shaikh)
and L.N.; Supervision, S.S. (Shehla Shaikh) and J.M.P.; Revision and changes S.S. (Salomi Shaikh),
L.N., S.S. (Shehla Shaikh) and J.M.P. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

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