Kaplan: Clinical Chemistry, 5th Edition

Clinical References - Methods of Analysis

Adrenocorticotropic Hormone (ACTH)

Hassan M.E. Azzazy i

Name: Adrenocorticotropic hormone (ACTH)

Molecular mass: ~4500 D
Chemical class: Polypeptide (39 amino acids)

{ED: “Refer to Chapter (number), (title), in the 5th edition of Clinical Chemistry: Theory,
Analysis, Correlation” missing here.}

Students’ Quick Hyperlink Review

• Principles of analysis and current usage
• Reference and preferred methods
• Specimen
• Interferences
• Reference Intervals
• Interpretation
• Performance goals
• References

Principles of Analysis and Current Usage

ACTH is a 39-amino-acid peptide, produced in the anterior pituitary as part of a large precursor
protein called pro-opiomelanocortin (POMC), which contains the sequence for several other
biologically active peptides as well. After secretion, POMC is rapidly cleaved to its component
peptides. The major stimulus for POMC (and hence ACTH) release is hypocortisolemia.

ACTH concentration can be determined by bioassay, receptor assays, or immunoassays.

Bioassays are based on the ability of ACTH to stimulate steroidogenesis or depletion of ascorbic
acid in isolated adrenocortical cells or in adrenal glands of hypophysectomized animals [1].
Bioassays are labor intensive and expensive. Receptor assays measure only the biologically
active fraction of ACTH [2]. These assays utilize solubilized binding proteins obtained from

i
Adrenocorticotropic Hormone (ACTH)
Previous and current authors of this method:
First edition: Not done
Methods edition: Barbara M. Goldsmith
Second edition: Not updated
Third edition: Not updated
Fourth edition: Not updated
Fifth edition: Hassan M.E. Azzazy

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

normal or neoplastic adrenocortical cells. Receptor assays suffer from several drawbacks,
including lability of receptor preparations and technical complexity.

Several radioimmunoassays (RIA) were initially developed for ACTH measurement. Many of
the RIA methods, however, required a pre-assay extraction step and extended incubation time
because they employed antibodies with low avidity, and the circulating concentration of ACTH
in healthy subjects is low [3,4].

Sandwich immunoradiometric assays (IRMA) have been shown to have several advantages over
the competitive immunoassays, including improved sensitivity, precision, and shorter incubation
time [5,6]. Rosano et al. [7] evaluated an IRMA developed for the measurement of ACTH in
human plasma. The assay employed goat polyclonal antibodies specific for ACTH 26-39 as
capture antibodies and 125I-labeled monoclonal antibodies specific for ACTH 1-17. Solid-phase
separation of the immune complex was achieved using mouse anti-goat antibody-coated
polystyrene beads. The assay had a detection limit of 1.7 ng/L and total imprecision of < 10% at
ACTH concentrations between 9 and 801 ng/L. A time-resolved immunofluorometric assay for
measurement of ACTH in unextracted plasma has also been reported [8].

Automated non-isotopic ACTH immunoassays with short incubation times have recently been
developed. The Nichols Advantage ACTH assay utilizes acridinium ester–labeled mouse
monoclonal antibody that binds to the C-terminal region of ACTH and a biotinylated goat
polyclonal antibody that binds to the N-terminal region.

After incubation with the patient’s plasma, streptavidin-coated magnetic beads are used to
separate the immune complex. Interassay imprecision at mean ACTH values of 17.7 and 731
ng/L were 12% and 6.1%, respectively [9]. A second assay, the DPC IMMULITE ACTH assay,
employs monoclonal murine antibodies coated on polystyrene beads and alkaline phosphatase–
labeled polyclonal rabbit anti-ACTH antibodies. This assay also uses a chemiluminescent
substrate. Interassay imprecision of this assay at mean ACTH concentration of 15.7 and 811 ng/L
were 11% and 2.8%, respectively. Additionally, a single-step electrochemiluminescence
immunoassay has been developed (Elecsys® ACTH assay). The assay utilizes two murine
monoclonal antibodies. One of the antibodies is labeled with ruthenium (detector), and the other
is a biotinylated antibody (capture) that binds to microparticles coated with streptavidin. The
reported assay detection limit is 0.5 ng/L, and the within-run and between-run imprecision (at
ACTH concentration 12 to 971 ng/L) were ≤1.9% and ≤5%, respectively [10].

Reference and Preferred Methods

There is no reference method for ACTH. Both isotopic and non-isotopic ACTH immunoassays
are reported to have comparable performance. Comparing results from different ACTH
immunoassays is possible only if calibrators are clearly defined. Two standards are currently
available from the National Institute of Biological Standards and Control (UK, purified human
ACTH 1-39; MRC 74/555; 6.2 IU/25 μg) and the National Pituitary Agency (Baltimore, MD;
synthetic ACTH 1-39; 4.71 IU/50 μg). IRMA ACTH assays are generally preferred over RIA for
investigating hypothalamic-pituitary-adrenal disorders. Because RIA assays utilize polyclonal
antibodies and will be able to recognize both fragments and whole-molecule ACTH, they are

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

recommended for investigating ectopic ACTH production by tumors, because tumors may
secrete fragments as well as intact ACTH.

Specimen
ACTH is unstable in blood, easily oxidized, strongly adsorbs to glass surfaces, and can be
rapidly degraded by plasma proteases into immunoreactive fragments. Therefore, proper care
must be taken in collection, handling, transportation, and storage of specimens. Preanalytical
variables such as time of day at which sample is collected, stress from a poorly performed
venipuncture, and/or prior administration of cortisol must be taken into consideration. Samples
for ACTH tests must be drawn prior to glucocorticoid administration. Morning (6:00 to 10:00
am) specimens are preferred. Blood is usually drawn in pre-chilled polystyrene EDTA tubes.
Plasma is separated immediately from cells in a refrigerated centrifuge and frozen within 15
minutes. Plasma is stored frozen at −20°C or preferably at −70°C in new plastic vials. For long-
term storage, aprotonin (500 kU/mL) should be added. Mercaptoethanol may be added to
specimens to protect ACTH against oxidation. Prior to assay, thawed plasma must be centrifuged
to remove any fibrin clots that may interfere with the assay.

Interferences
ACTH concentration follows a nycthemeral rhythm, with highest levels observed between 6:00
and 8:00 a.m. and lowest levels between 9:00 and 10:00 p.m. For proper monitoring, samples
should be collected at the same time each day (early morning preferred). Pregnancy,
menstruation, and stress may increase ACTH secretion. Aminoglutethimide, amphetamine,
levodopa, metoclopramide, metyrapone, pyrogens, RU-486, vasopressin, hypoglycemia, and
insulin have been reported to increase plasma ACTH levels. Depressed ACTH levels may be
observed following administration of dexamethasone or other corticosteroids or collection of
specimens in tubes containing heparin.

Heterophilic antibodies in serum of humans routinely exposed to animals or animal serum

products can react with the immunoglobulins included in ACTH immunoassays, causing
interference [11].

Reference Intervals
Method Specimen Reference Range (pg/mL)
a
RIA EDTA plasma Newborn (1 day) 10-185
b
Adult (8:00 am) <120
b
Adult (4:00-8:00 pm) <85
a
[Endocrine Sciences. Pediatric
Laboratory Services. Tarzana, CA:
Endocrine Sciences; 1988.]
b
[Vanderbilt Pathology Laboratory
Services. Test Catalog. Nashville,
TN: Vanderbilt Pathology Laboratory
Services; 1989.]
IRMA EDTA plasma Adult (7:00-10:00 am) 9-52
[Nichols Institute Reference

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

 Laboratories. Test Catalog. San Juan
Capistrano, CA: Nichols Institute
Research Laboratories; 1993.]
RIA Amniotic fluid 10-18 weeks: 209
26-30 weeks: 430
35-36 weeks: 162
[Tulchinsky D, Ryan KJ, eds. Maternal-
Fetal Endocrinology. Philadelphia:
Saunders; 1980.]
Automated EDTA plasma Adult (AM draws) 10-60
chemiluminescent
immunometric [Mayo Clinic. Test Catalog. Rochester,
assay MN: Mayo Clinic; 2005.]

Interpretation
Determination of ACTH in plasma is the best test to distinguish between primary and secondary
adrenal insufficiency. Measurement of plasma ACTH concentration is used to assess Cushing’s
disease, adrenal tumors, ectopic ACTH-producing tumors, Addison’s disease, Nelson’s
syndrome, adrenal tumors, and hypopituitarism. ACTH secretion is pulsatile and increases in
cases of hypocortisolemia when the pituitary and hypothalamus are intact.

In primary adrenal insufficiency, ACTH levels usually exceed 100 pg/mL by RIA. It should be
noted, however, that ACTH level alone cannot be used to differentiate between control subjects
and patients with central hypoadrenalism. One study reported identical ACTH levels in control
subjects (4 to 81 pg/mL) and patients with confirmed pituitary disease (8 to 75 pg/mL) [12].

In Addison’s disease (primary adrenal insufficiency), elevated ACTH levels (>1000 pg/mL) are
typical. ACTH levels are also elevated in congenital adrenal hyperplasia, pituitary-dependent
Cushing’s disease, ectopic ACTH-producing tumors, and Nelson’s syndrome. ACTH
determinations can also help to identify the cause of cortisol hypersecretion in Cushing’s
syndrome. ACTH levels are typically low when this is due to lesions or hyperplasia of the
adrenal cortex, and high in cases of ectopic ACTH production or hypersecretion of ACTH by the
pituitary. Low levels are observed when adrenal insufficiency is secondary to pituitary
dysfunction, adrenal carcinoma, adenoma, and hypopituitarism.

Because the major stimulus to ACTH production is hypocortisolemia, interpretation of ACTH

concentration requires concurrent measurement of cortisol to be made. High levels of ACTH and
cortisol are observed in Cushing’s disease and ectopic ACTH secretion. Low levels of both
hormones are observed in hypopituitarism. Elevated levels of cortisol with low ACTH may
indicate adrenal tumor. Elevated ACTH and low cortisol levels are observed in Addison’s
disease.

ACTH in amniotic fluid may be of fetal origin, and low levels may be observed in anencephalic
fetuses; high levels may indicate immaturity of hypothalamic regulation. Dexamethasone
suppression testing may be useful for distinguishing causes of increased ACTH levels. High-

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

Clinical References - Methods of Analysis 2-5

dose dexamethasone suppresses ACTH and cortisol secretion in Cushing’s disease but has no
effect in cases of adrenal adenomas, adrenal carcinomas, and ectopic ACTH producing tumors.

Performance Goals
The current Clinical Laboratory Improvement Amendments (CLIA) performance goal for
measurement of ACTH is for laboratories to be within ±3 standard deviations of the peer-group
mean. According to the 2007 College of American Pathologists (CAP) Survey, coefficients of
variation (CV) for all ACTH methods at mean values of 168.6 pg/mL (SD 20.6) and 311.3
pg/mL (SD 45.8) were 12.2% and 14.7%, respectively.

References
1 Sayers G. Bioassay of ACTH using isolated cortex cells. Applications: structure activity
relationship for ACTH and analogues, assay of corticotrophin-releasing factor, and assay of
plasma ACTH. Ann NY Acad. Sci 1977;297:220-41.
2 Lefkowitz RJ, Roth J, Pastan I. Radioreceptor assay of adrenocorticotropic hormone: new
approach to assay of polypeptide hormones in plasma. Science 1970;170:633-5.
3 Gutkowska J, Julesz J, St-Louis J, Genest J. Radioimmunoassay of corticotropin from
plasma. Clin Chem 1982;28:2229-34.
4 Arts CJ, Koppeschaar HP, Veeman W, Thijssen JH. A direct radioimmunoassay for the
determination of adrenocorticotropic hormone (ACTH) and a clinical evaluation. Ann Clin
Biochem 1985;22:247-56.
5 Dobson SH, Gray C, Smith H, Baker T, Ratcliffe JG, White A. Selection and optimization
of monoclonal antibodies for a two-site immunoradiometric assay for ACTH. J Immunol
Methods 1986;88:83-90.
6 Zahradnik R, Brennan G, Hutchison JS, Odell WD. Immunoradiometric assay of
corticotropin with use of avidin-biotin separation. Clin Chem 1989;35:804-7.
7 Rosano TG, Demers LM, Hillam R, Dybas MT, Leinung M. Clinical and analytical
evaluation of an immunoradiometric assay for corticotropin. Clin Chem 1995;41:1022-7.
8 Dobson S, White A, Hoadley M, Lovgren T, Ratcliffe J. Measurement of corticotropin in
unextracted plasma: comparison of a time-resolved immunofluorometric assay and an
immunoradiometric assay, with use of the same monoclonal antibodies. Clin Chem
1987;33:1747-51.
9 Vogeser M, Engelhardt D, Jacob K. Comparison of two automated adrenocorticotropic
hormone assays. Clin Chem 2000;46:1998-2000.
10 Verschraegen I, Anckaert E, Schiettecatte J, Mees M, Garrido A, Hermsen D et al.
Multicenter evaluation of a rapid electrochemiluminescent adrenocorticotropic hormone
(ACTH) immunoassay. Clin Chim Acta 2007;380:75-80.
11 Boscato LM, Stuart MC. Heterophilic antibodies: a problem for all immunoassays. Clin
Chem 1988:34:27- 33.
12 Oelkers W, Diederich S, Bahr V. Diagnosis and therapy surveillance in Addison’s disease:
rapid adrenocorticotropin (ACTH) test and measurement of plasma ACTH, renin activity,
and aldosterone. J Clin Endocrinol Metab 1992;75:259-64.

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.