Laboratory Diagnosis

of Adrenocortical Diseases

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OBJECTIVES:
By the end of this session the student should
be able to:
Discuss the adrenal-pituitary-hypothalamus axis, including
relationships between hormones and steroids in each
Discuss the use of serum cortisol in the workup of
hypercortisolism and hypocortisolism
Discuss the use of urinary free cortisol in the workup of
hypercortisolism
Describe the use of serum ACTH in differentiating primary from
secondary hypocortisolism, in determining the cause of Cushing's
disease, in demonstrating ectopic ACTH production, and in the
diagnosis of congenital adrenal hyperplasia
Discuss the performance of the short, low-dose, and high-dose
dexamethasone suppression test and the interpretation of test results
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 Describe the short and the long ACTH stimulation tests and their use in
distinguishing primary from secondary hypocortisolism
Discuss the corticotrophin-releasing hormone stimulation test and its use to
distinguish Cushing's disease and ectopic ACTH production
Describe the metapyrone test and its use in monitoring the intactness of the pituitary-
adrenal axis
Discuss congenital adrenal hyperplasia and the findings with 21-hydroxylase
deficiency (no cortisol, no aldosterone, ambiguous genitalia, elevation of serum
17-α-hydroxyprogesterone)
Describe primary hyperaldosteronism (Conn's syndrome) and its causes (bilateral
adrenal hyperplasia and adrenal adenoma)
Describe serum aldosterone and renin in primary hyperaldosteronism
Discuss secondary hyperaldosteronism and its causes (cirrhosis, nephrotic syndrome,
congestive heart failure)
Describe serum aldosterone and renin in secondary hyperaldosteronism
Discuss the signs and symptoms of, and lab tests used for, hypercortisolism
Discuss the signs and symptoms of, and lab tests used for, hypocortisolism
Describe changes in serum electrolytes, glucose, and urea-nitrogen in
hypercortisolism
Describe changes in serum electrolytes, glucose, and urea-nitrogen in
hypocortisolism 3
The diagnostic approach to the following adrenocortical conditions

is discussed below:

 Hypercortisolism

 Hypocortisolism

 Hyperaldosteronism

 Congenital adrenal hyperplasia

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The following tests serve in the evaluation of the above conditions:

1. Serum cortisol
2. Urinary free cortisol
3. ACTH in plasma
4. Dexamethasone suppression test
5. ACTH stimulation test
6. CRH stimulation test
7. Metyrapone test
8. Serum 17-α-hydroxyprogesterone
9. Diagnosis of 21-Hydroxylase Deficiency
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 1- Serum Cortisol
- Cortisol is produced in the zona fasciculata of the
adrenal cortex from cholesterol through a series of
enzymatic reactions, the rates of which are controlled by
ACTH.

- Cortisol serum levels are lowest from midnight to 4

a.m and highest from 6 to 9 a.m. However, for
practical reasons, sampling of blood for studying the
diurnal rhythm is done at 8 a.m. and 4 p.m., the
evening values normally being about half of the
morning values. .
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 Transcortin normally is nearly saturated with bound cortisol.
Thus, as in hypercortisolism, the binding capacity of CBG is
exceeded when cortisol levels rise beyond 20 to 25 μg/dL,
resulting in a steep rise in free cortisol. The result is a sharp
increase in urinary excretion of free cortisol.

 Reference Ranges: Morning values: 6 - 24 μg/dL

Evening values: 3 - 12 μg/dL

 Interpretation of abnormal values

Increased values: are seen in Cushing’s syndrome,
alcoholism, stress, and in starvation.
Low values: are seen in Addison’s disease, chronic
illnesses, and in adrenogenital syndrome.

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 2- URINARY FREE CORTISOL

 As the urinary excretion of cortisol parallels the

level of free plasma cortisol, the former can be
used as an indirect measure of the latter.
 A rise in plasma cortisol beyond about 20 μg/dL,
approximate saturation level of transcortin occur,
leading to a sharp increase of free cortisol and
urinary cortisol excretion.
 For instance, a rise of plasma cortisol from 20 to 25 μg/dL may be
accompanied by a doubling of urinary free cortisol, or a rise from 20
to 40 μg/dL in plasma may be accompanied by a five-fold rise in the
urine.
 In the diagnosis of Cushing’s syndrome, the sensitivity of this test is
96.9% and the specificity is 94.3%. Values seen in Cushing’s
syndrome are almost always higher than 120 μg/day.
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 Furthermore, in conditions of increased turnover of corticosteroids,
as in obesity, or increases in transcortin, as in pregnancy, where the
level of circulating free cortisol is not increased, the urinary free
cortisol values remain normal.

- This test is not useful in the assessment of Addison’s disease, since

a great deal of overlap of values occurs in the lower range of normal.

- Reference Range: 20-90 μg/day

- Precautions: a careful 24 hour urine collection is necessary; values
may be low in renal disease with reduced glomerular filtration;
determination of 24 hour urine creatinine excretion is of help to
evaluate complete urine collection (1-2 g/24 hour)
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 3-ADRENOCORTICOTROPHIC HORMONE (ACTH)

 ACTH is very unstable in blood

samples because of its destruction
by proteolytic enzymes within
a few minutes.

 Test (specimens) conditions are rigidly controlled (addition of

proteolysis inhibitors, immediate icing of blood samples, immediate
handling by the laboratory during standard sampling times between 8
and 10 a.m, centrifuged at 4°C and immediately frozen.).

 It is adsorbed onto glass and thus glass collection tubes should be

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avoided.
 Reference Range :
 50 to 100 pg/mL ( if detects intact molecules and some
fragments as in detection of ectopic tumors).
 10-50 pg/mL (if detects only intact ACTH as in evaluating
pituitary ACTH secretion).

 The test is used

1. To differentiate primary from secondary adrenal insufficiency.
2.To determine the cause of Cushing’s syndrome.
3.In the demonstration of ectopic ACTH production by rapidly
growing malignancies before the full-blown Cushing’s syndrome
develops.
4.In the diagnosis of congenital adrenal hyperplasia.
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 4- DEXAMETHASONE SUPPRESSION TEST

• This test is used in the differential diagnosis

of Cushing’s syndrome either pituitary-
dependent or not.

• Dexamethasone
• Is a synthetic fluorine-containing cortisol
analogue which has 10 to 20 times the metabolic potency of cortisol,
including its negative feedback action on the pituitary.
• When given to patients in small test doses, dexamethasone inhibits
ACTH release from the pituitary without contributing
measurably to the total pool of glucocorticoids. Thus, under these
conditions, dexamethasone does not interfere with plasma cortisol
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and urinary steroid measurements.
Three versions of this test

A) The short (overnight) dexamethasone suppression test

This is a simple screening test only used to rule out Cushing’s.
Procedure :
One (or two) mg of dexamethasone is given orally to the patient at 11:00
p.m., and at 8:00 a.m. a blood specimen is obtained for serum cortisol
measurement.
Interpretation of results
- Serum cortisol less than 5 μg/dL = normal
- Serum cortisol greater than 10 μg/dL = possible Cushing’s syndrome
Advantages
Simplicity; may be done on an outpatient basis
Disadvantages
False positives (nonsuppression) in psychiatric disease, stress, high
cortisol values because of increased transcortin (pregnancy,
contraception) and obesity.
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False negatives are rare (< 1%).
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B) The low-dose dexamethasone suppression test
The purpose of this test is the same as the short (overnight) test, but
there are fewer false positives with this test. However, there are more
false negatives (suppression in true Cushing’s) with this test than
with the short test above.
Procedure
A 0.5 mg dose of dexamethasone is given orally every 6 hours for 48
hours
Interpretation of results
- Serum cortisol less than 5 μg/dL = normal
- Serum cortisol greater than 10 μg/dL = Cushing’s syndrome
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C) The high-dose dexamethasone suppression test
This test is performed as a follow-up after a low-dose test has shown
no suppression. Its purpose is to distinguish adrenal hyperplasia
caused by tumors of the pituitary (basophilic adenomas) from
autonomous cortisol-producing adrenal adenomas and
carcinomas, and ectopic carcinomas (i.e., oat cell carcinoma of the
lung). The test is also helpful in distinguishing Cushing’s disease
from ectopic ACTH secretion; the ectopic tumor generally resists
negative feedback.
Procedure :
24 hour urine collections are obtained daily for 4 days for free cortisol
and 17-hydroxycorticosteroid determinations; Dexamethasone, 2.0 mg
orally every six hours, is begun at 8 am on the second day and
continued for eight doses; free cortisol and creatinine are measured in
each twenty-four hour urine sample; other measurements include
plasma cortisol at 8 a.m. and 8 p.m. on the first day to look for diurnal
variation and at 8 a.m. on the fifth day.
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Interpretation of results
- Patients with Cushing’s disease due to an ACTH-secreting pituitary
adenoma usually show suppression of urinary free cortisol and 17-
hydroxycorticosteroid excretion greater than or equal to 50% of
baseline by the fourth day; lack of diurnal variation in plasma cortisol;
and plasma cortisol ≤10 μg/dL at 8 a.m. on the fifth day.

- Urinary free cortisol not suppressed = either autonomous production

of cortisol due to adrenocortical adenoma or carcinoma or
autonomous ACTH production ectopically by certain types of
carcinoma.

- Ectopic ACTH production sometimes can be demonstrated by selective

venous blood sampling (via catheterization) from blood vessels
draining tumor areas.
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 5- ACTH STIMULATION TEST

- The purpose of this test is to

assess the ability of the
adrenal cortex to respond to
ACTH stimulation by
production of glucocorticoids.
- The test is used mostly in the differentiation of primary
hypocortisolism (due to adrenal failure, i.e. Addison’s disease) from
secondary hypocortisolism due to hypopituitarism.
- ACTH is a polypeptide chain composed of 39 amino acids. The
biological activity of the hormone resides in that portion of the
molecule comprising amino acid residues 1-24 (identical in al
species so far examined). For medical use a synthetic ACTH
consisting only of these 24 amino acids has been prepared. It has full
biological activity and lacks antigenicity. The preparation mostly use
in the U.S. is Cortrosyn. A short and a long version of the test are29 in
use.
The short ACTH stimulation test

Procedure:
• A blood specimen is first drawn to
obtain a baseline cortisol and then
250 μg of Cortrosyn are given IV.
Thirty and sixty minutes later,
additional blood specimens are drawn
for the stimulated cortisol levels.

Interpretation of results

• A normal response is a rise of at least 7 μg/dL after 30 minutes and a

rise of 11 μg/dL after 60 minutes.
• If a subnormal response or no response is observed, the long
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stimulation test is indicated.
The long ACTH stimulation test

• Intravenous infusions of ACTH over

6 to 8 hours are given for 2 to 5 days.
Urine free cortisol and serum cortisol are
measured daily.

Interpretation of results
• A progressive staircase rise is seen over 2 to 3 days in adrenal
insufficiency caused by pituitary or hypothalamic disease or steroid
level suppression.
• Little or no increases in cortisol secretion are seen in primary adrenal
failure as in congenital adrenal hyperplasia due to 21- and 17-
hydroxylase deficiencies. 31
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 6- CRH STIMULATION TEST

 The CRH (corticotrophin releasing hormone) stimulation test may

help to differentiate between Cushing’s disease and ectopic ACTH
production.
 An intravenous bolus (100 μg) of CRH results in an increase in
plasma cortisol levels in normal individuals and patients with tertiary
adrenal insufficiency (hypothalamic).

 Most patients with Cushing’s disease show normal or, frequently,

exaggerated ACTH and cortisol responses to synthetic CRH, whereas
patients with ectopic ACTH-secreting or adrenocortical tumors show
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no response
 7- METYRAPONE TEST
• Metyrapone selectively inhibits 11 β-hydroxylase in the adrenal cortex. This enzyme
is involved in the conversion of 11-desoxycortisol (Compound S) to cortisol, the last
step in the formation of cortisol. Thus, when metyrapone is given to a patient, cortisol
production is inhibited.

This has two consequences:

• The negative feedback on the pituitary by cortisol is diminished, and the normal
pituitary responds with an increase of ACTH release. The increased ACTH stimulates
the adrenal cortex to produce adrenocortical hormones (except cortisol, whose
synthesis is blocked) and to increase production of the precursors of cortisol (e.g.,
Compound S or 11-desoxycortisol) which appear in the circulating blood and in the
urine, where they can be measured.

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• In clinical practice metyrapone is used to test the ability of
the pituitary to produce ACTH and to check the intactness
of the pituitary- adrenal axis.

• The test differentiates only between normal and adrenal insufficiency.

Since both a normal pituitary and a normal adrenal are required for a
normal response, the test cannot differentiate between primary and
secondary adrenal insufficiency.

• Furthermore, since metyrapone accentuates a pre-existing

hypocortisolism, an acute crisis of adrenal insufficiency may be
provoked by the test. For these reasons as well as the length of the test
requiring hospitalization for several days, the metyrapone test is used
infrequently.

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 8- SERUM 17-α-HYDROXYPROGESTERONE

 This test is used in the differential diagnosis of adrenogenital

syndrome (congenital adrenal hyperplasia) to detect 21-hydroxylase
deficiency.
 This enzyme converts 17-α-hydroxyprogesterone to 11-desoxycortisol
in the pathway of cortisol biosynthesis in the adrenal.
 If the enzyme is absent, this biosynthetic step is blocked and 17-α-
hydroxyprogesterone accumulates in high concentration in the blood.
 This condition may constitute a life-threatening medical emergency.
 The test can be done within a few hours (without having to resort to
24-hour urine collections) before steroid treatment is instituted.
Reference values
 Less than 100 ng/dL
 In patients with 21-hydroxylase deficiency: greater than 1000 ng/dL

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 9- Diagnosis of 21-Hydroxylase Deficiency

 Types: A clinically severe and a milder form of this disease are

observed.

 In the severe form:

 There is marked sodium depletion since the 21-hydroxylase not

only is involved in the biosynthesis of cortisol but also in that of
aldosterone. Thus, these infants suffer from hypocortisolism and
hypoaldosteronism with salt loss.
 Because the negative feedback control by cortisol on the pituitary
is absent, ACTH production is unchecked and the precursors prior
to the enzymatic block accumulate. These precursors lead to
increased production of androgens that are the causes of the
anomalous genital development, virilization
(pseudohermaphroditism) in girls, and precocious puberty in40boys.
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 In the clinically severe form:
 Correction of the electrolyte abnormality and steroid
replacement should be initiated without delay.
Blood and urine samples should also be collected prior to or at
the beginning of treatment. Serum 17-α-hydroxyprogesterone
may be elevated more than 10-fold, which is virtually diagnostic.

 If successful replacement steroid therapy is instituted, ACTH

production by the pituitary is inhibited, and the abnormally high
levels of serum 17-α-hydroxyprogesterone and urinary steroids
decrease. This can be used as a guide to successful treatment.
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 In the milder form :

 The major abnormality is hypocortisolism while sufficient

aldosterone is produced to prevent salt loss. However, virilization
is still seen because of the overproduction of androgens.
 In some patients, the only biochemical abnormality is an elevated
serum level of 17-α-hydroxyprogesterone which is the diagnostic
feature of this condition. These patients may be capable of
producing sufficient cortisol for normal maintenance but are unable
to increase cortisol when under stress.

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