Adrenal Function Tests

Introduction

• The adrenal glands are small, triangular-shaped endocrine organs located on top of each kidney.
• Despite their small size, these glands play a crucial role in maintaining homeostasis by producing hormones that regulate metabolism, blood pressure, electrolyte balance, immune function, stress responses, and sexual development.

The adrenal gland consists of two anatomically and functionally distinct regions:

1. Adrenal Cortex
   • Zona glomerulosa: Produces aldosterone
   • Zona fasciculata: Produces cortisol
   • Zona reticularis: Produces adrenal androgens
2. Adrenal Medulla
   • Produces catecholamines (epinephrine and norepinephrine)

Disorders of adrenal function can lead to significant morbidity and even life-threatening emergencies. Therefore, laboratory assessment of adrenal hormones forms an essential component of endocrine diagnostics.

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Anatomy and Physiology of the Adrenal Gland

Before discussing adrenal function tests, it is important to understand the hormones secreted by the adrenal gland.

Hormones of the Adrenal Cortex

1. Cortisol

Cortisol is the major glucocorticoid hormone.

Functions:

• Regulation of glucose metabolism
• Protein and fat metabolism
• Anti-inflammatory effects
• Stress response
• Maintenance of blood pressure

Its secretion is controlled by the hypothalamic-pituitary-adrenal (HPA) axis:

Hypothalamus → CRH → Pituitary → ACTH → Adrenal Cortex → Cortisol

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2. Aldosterone

Aldosterone is the principal mineralocorticoid.

Functions:

• Sodium retention
• Potassium excretion
• Regulation of blood pressure
• Maintenance of extracellular fluid volume

Its secretion is mainly regulated by:

• Renin-Angiotensin-Aldosterone System (RAAS)
• Serum potassium levels

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3. Adrenal Androgens

Major hormones include:

• Dehydroepiandrosterone (DHEA)
• DHEA-Sulfate (DHEAS)
• Androstenedione

Functions:

• Development of secondary sexual characteristics
• Pubic and axillary hair growth
• Libido in females

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Hormones of the Adrenal Medulla

The adrenal medulla produces:

• Epinephrine (adrenaline)
• Norepinephrine (noradrenaline)

Functions:

• Fight-or-flight response
• Increased heart rate
• Elevated blood pressure
• Enhanced glucose availability

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Indications for Adrenal Function Testing

Adrenal testing is indicated in patients presenting with:

Symptoms of Adrenal Insufficiency

• Chronic fatigue
• Weight loss
• Hypotension
• Hyperpigmentation
• Hyponatremia

Symptoms of Cushing Syndrome

• Central obesity
• Moon face
• Buffalo hump
• Purple striae
• Hypertension

Symptoms of Hyperaldosteronism

• Resistant hypertension
• Muscle weakness
• Hypokalemia

Symptoms of Pheochromocytoma

• Episodic hypertension
• Palpitations
• Excessive sweating
• Headache

Features of Androgen Excess

• Hirsutism
• Virilization
• Menstrual disturbances
• Infertility

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Classification 

Adrenal function tests can be divided into:

Basal Hormone Measurements

• Serum cortisol
• Plasma ACTH
• Aldosterone
• Renin activity
• DHEAS
• Catecholamines

Dynamic Function Tests

• ACTH stimulation test
• Dexamethasone suppression test
• CRH stimulation test
• Insulin tolerance test

Urinary Hormone Measurements

• 24-hour urinary free cortisol
• Urinary catecholamines
• Urinary metanephrines

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Cortisol Assessment

Serum Cortisol

• Serum cortisol is one of the most commonly requested adrenal function tests.

Normal Circadian Rhythm

Cortisol secretion follows a diurnal pattern:

• Highest: 6–8 AM
• Lowest: Midnight

Therefore, sample timing is critical.

Clinical Applications

Low Cortisol Levels

Suggest:

• Addison disease
• Secondary adrenal insufficiency
• Pituitary disorders

Elevated Cortisol Levels

Suggest:

• Cushing syndrome
• Stress
• Severe illness

Reference Values

Morning cortisol:

• Approximately 5–25 μg/dL

Values vary among laboratories.

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Plasma Free Cortisol

Only about 5–10% of circulating cortisol exists in the free biologically active form.

Most cortisol is bound to:

• Cortisol-binding globulin (CBG)
• Albumin

Measurement of free cortisol may be useful when CBG levels are altered.

Examples include:

• Pregnancy
• Estrogen therapy
• Liver disease

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Salivary Cortisol Assessment

• Salivary cortisol reflects free cortisol concentration and is increasingly used in endocrine practice.

Advantages

• Non-invasive
• Easy sample collection
• Convenient for home testing
• Reflects biologically active cortisol

Clinical Application

Particularly useful for:

• Screening Cushing syndrome
• Assessing circadian rhythm abnormalities

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Late-Night Salivary Cortisol

• Normally, cortisol levels are very low at midnight.
• Elevated Midnight Cortisol

Strongly suggests:

• Cushing syndrome
• Loss of normal circadian rhythm

Advantages

• High sensitivity
• High specificity
• Convenient outpatient testing

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Urinary Free Cortisol (UFC)

• Twenty-four-hour urinary free cortisol is an important test for diagnosing cortisol excess.

Principle

• Free cortisol is filtered through the glomerulus and excreted in urine.
• Measurement reflects total daily cortisol production.

Procedure

• Complete 24-hour urine collection
• Proper storage and preservation
• Laboratory quantification

Clinical Significance

Elevated UFC

Occurs in:

• Cushing disease
• Adrenal adenoma
• Adrenal carcinoma
• Ectopic ACTH syndrome

Advantages

• Reflects integrated cortisol secretion over 24 hours
• Eliminates circadian variation

Limitations

• Incomplete urine collection may affect results
• Renal impairment may alter excretion

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Plasma ACTH Measurement

Biological Characteristics of ACTH

Structure

• Peptide hormone consisting of 39 amino acids
• Derived from proopiomelanocortin (POMC)

Half-Life

ACTH has a very short plasma half-life of approximately:

• 8–12 minutes

Because of its rapid degradation, careful specimen handling is essential.

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Circadian Rhythm of ACTH

• Like cortisol, ACTH secretion follows a circadian rhythm.

Peak Levels

Highest concentrations occur:

• Between 6:00 AM and 8:00 AM

Lowest Levels

Lowest concentrations occur:

• Around midnight

ACTH secretion also occurs in a pulsatile manner throughout the day.

For accurate interpretation, ACTH samples are typically collected in the early morning.

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Indications for Plasma ACTH Measurement

ACTH measurement is useful in patients with:

• Suspected Adrenal Insufficiency

Symptoms include:

• Chronic fatigue
• Weakness
• Weight loss
• Hypotension
• Hyperpigmentation

Suspected Cushing Syndrome

Features include:

• Central obesity
• Moon face
• Buffalo hump
• Purple striae
• Hypertension

Pituitary Disorders

• Hypopituitarism
• Pituitary adenoma
• ACTH deficiency

Adrenal Tumors

• Functional adrenal adenoma
• Adrenal carcinoma

Ectopic ACTH Syndrome

ACTH-producing tumors such as:

• Small-cell lung carcinoma
• Bronchial carcinoid tumors

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Sample Collection and Handling

• ACTH is highly unstable in blood samples; therefore, strict pre-analytical precautions are required.

Specimen Type

• Plasma collected in EDTA tubes

Collection Procedure

1. Blood should be drawn into a chilled EDTA tube.
2. Sample should be transported on ice.
3. Plasma should be separated immediately.
4. Frozen plasma is preferred for delayed analysis.

Methods of ACTH Measurement

Several laboratory techniques are available.

• Immunoradiometric Assay (IRMA)
• Chemiluminescent Immunoassay (CLIA)
• Electrochemiluminescence Immunoassay (ECLIA)

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ACTH Stimulation Test (Synacthen Test)

• The ACTH Stimulation Test is the most commonly used dynamic endocrine test for evaluating adrenal cortical function and diagnosing adrenal insufficiency.
• It assesses the ability of the adrenal cortex to produce cortisol in response to stimulation by synthetic ACTH (cosyntropin or Synacthen).

Principle

Administration of synthetic ACTH should stimulate the adrenal glands to secrete cortisol. Healthy adrenal glands respond with a significant increase in serum cortisol levels, whereas damaged or atrophic adrenal glands show little or no response.

Procedure

1. Measure baseline serum cortisol.
2. Administer 250 μg synthetic ACTH intravenously or intramuscularly.
3. Measure serum cortisol at 30 and/or 60 minutes after injection.

Interpretation

Normal Response

• Significant rise in serum cortisol concentration.
• Peak cortisol generally >18–20 μg/dL (500–550 nmol/L).

Abnormal Response

• Minimal or absent increase in cortisol levels.
• Suggests primary adrenal insufficiency (Addison disease) or chronic adrenal atrophy.

Clinical Uses

• Diagnosis of primary adrenal insufficiency
• Evaluation of suspected adrenal failure
• Assessment of adrenal reserve after prolonged glucocorticoid therapy
• Investigation of hypothalamic-pituitary-adrenal (HPA) axis dysfunction

Advantages

• Simple and safe procedure
• Rapid results
• High diagnostic value for adrenal insufficiency
• Widely used in clinical endocrinology

Limitation

The test may be normal in recent-onset secondary adrenal insufficiency because the adrenal glands may still retain responsiveness to ACTH.

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Dexamethasone Suppression Test

• The Dexamethasone Suppression Test (DST) is an important dynamic endocrine test used to evaluate hypercortisolism and diagnose Cushing syndrome.
• It assesses the integrity of the hypothalamic-pituitary-adrenal (HPA) axis by determining whether cortisol secretion can be suppressed by dexamethasone, a potent synthetic glucocorticoid.

Principle

Dexamethasone suppresses the secretion of Adrenocorticotropic Hormone (ACTH) from the pituitary gland through negative feedback. In healthy individuals, this leads to a reduction in cortisol production by the adrenal cortex. Patients with Cushing syndrome fail to suppress cortisol adequately.

Overnight Low-Dose Dexamethasone Suppression Test

Procedure

1. Administer 1 mg dexamethasone orally at 11:00 PM.
2. Measure serum cortisol at 8:00 AM the following morning.

Interpretation

Normal Response

• Morning serum cortisol is suppressed to <1.8 μg/dL (50 nmol/L).
• Indicates normal HPA axis function.

Abnormal Response

• Cortisol remains elevated despite dexamethasone administration.
• Suggests Cushing syndrome or autonomous cortisol secretion.

Clinical Uses

• First-line screening test for Cushing syndrome
• Evaluation of unexplained obesity, hypertension, and diabetes associated with cortisol excess
• Assessment of adrenal and pituitary causes of hypercortisolism

Advantages

• Simple and convenient outpatient test
• High sensitivity for detecting cortisol excess
• Widely used as an initial screening investigation

Limitations

False-positive results may occur in:

• Severe stress or illness
• Depression
• Alcohol dependence
• Obesity

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Evaluation of Cushing Syndrome

The diagnosis usually begins with one of the following:

1. Late-Night Salivary Cortisol

Advantages:

• Non-invasive
• Convenient
• Reflects free cortisol

Loss of normal midnight cortisol suppression suggests Cushing syndrome.

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2. 24-Hour Urinary Free Cortisol

Measures biologically active cortisol.

Elevated levels indicate:

• Cushing disease
• Adrenal tumors
• Ectopic ACTH production

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Aldosterone and Renin Testing

Indications for Testing

Aldosterone and renin measurements are indicated in patients with:

• Resistant or severe hypertension
• Unexplained hypokalemia
• Adrenal incidentalomas
• Early-onset hypertension
• Family history of primary hyperaldosteronism
• Suspected mineralocorticoid excess

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Aldosterone Measurement

Plasma aldosterone concentration (PAC) is measured using immunoassay or LC-MS/MS methods.

Elevated Aldosterone Levels

May occur in:

• Primary hyperaldosteronism (Conn syndrome)
• Bilateral adrenal hyperplasia
• Aldosterone-producing adenoma
• Secondary hyperaldosteronism

Reduced Aldosterone Levels

May occur in:

• Addison disease
• Hyporeninemic hypoaldosteronism
• Congenital adrenal disorders

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Plasma Renin Activity (PRA)

Renin can be measured as:

• Plasma Renin Activity (PRA)
• Direct Renin Concentration (DRC)

Elevated Renin

Observed in:

• Renal artery stenosis
• Congestive heart failure
• Dehydration
• Secondary hyperaldosteronism

Reduced Renin

Observed in:

• Primary hyperaldosteronism
• Excess mineralocorticoid states

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Aldosterone-to-Renin Ratio (ARR)

The Aldosterone-to-Renin Ratio (ARR) is the preferred screening test for primary hyperaldosteronism.

Calculation

ARR = Plasma Aldosterone Concentration ÷ Plasma Renin Activity

Interpretation

High ARR

Characterized by:

• Elevated aldosterone
• Suppressed renin

Strongly suggests:

• Primary hyperaldosteronism (Conn syndrome)

Normal or Low ARR

Suggests:

• Normal RAAS function
• Secondary causes of hypertension

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Factors Affecting Results

Several factors can influence aldosterone and renin levels:

Physiological Factors

• Body posture
• Dietary sodium intake
• Time of sample collection
• Pregnancy

Medications

• Diuretics
• ACE inhibitors
• Angiotensin receptor blockers (ARBs)
• Beta blockers
• Spironolactone
• Eplerenone

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Saline Suppression Test

The Saline Suppression Test (SST) is a confirmatory test used to diagnose primary hyperaldosteronism (Conn syndrome) in patients who have an elevated Aldosterone-to-Renin Ratio (ARR). The test evaluates whether aldosterone secretion can be suppressed by sodium loading.

Principle

In healthy individuals, intravenous administration of saline expands the extracellular fluid volume, suppresses renin release, and consequently reduces aldosterone secretion. In primary hyperaldosteronism, aldosterone production is autonomous and remains elevated despite saline infusion.

Procedure

1. The patient remains in a recumbent position.
2. Baseline plasma aldosterone concentration is measured.
3. 2 liters of 0.9% normal saline are infused intravenously over 4 hours.
4. Plasma aldosterone is measured at the end of the infusion.

Interpretation

Normal Response

• Aldosterone secretion is suppressed after saline loading.
• Post-infusion plasma aldosterone concentration typically falls to <5 ng/dL.

Abnormal Response

• Aldosterone remains elevated despite saline infusion.
• Post-infusion aldosterone concentration >10 ng/dL strongly suggests primary hyperaldosteronism.

Clinical Uses

• Confirmation of primary hyperaldosteronism
• Evaluation of patients with elevated ARR
• Differentiation of autonomous aldosterone secretion from secondary causes of hypertension

Advantages

• Widely accepted confirmatory test
• Relatively simple to perform
• Good diagnostic accuracy

Limitations

The test should be used cautiously or avoided in patients with:

• Severe uncontrolled hypertension
• Congestive heart failure
• Significant renal impairment
• Severe hypokalemia

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Adrenal Androgen Assessment

Physiological Role

Adrenal androgens contribute to:

• Development of pubic and axillary hair
• Maintenance of libido, particularly in females
• Peripheral synthesis of sex hormones
• Secondary sexual characteristics

Their secretion is primarily regulated by Adrenocorticotropic Hormone (ACTH).

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Major Adrenal Androgens

1. Dehydroepiandrosterone (DHEA)

• Weak androgen produced by the adrenal cortex
• Precursor for testosterone and estrogen synthesis

2. Dehydroepiandrosterone Sulfate (DHEAS)

• Sulfated form of DHEA
• Most abundant circulating adrenal androgen
• Longer half-life and more stable serum concentration
• Preferred marker of adrenal androgen production

3. Androstenedione

• Intermediate steroid hormone
• Precursor of testosterone and estrone

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Indications for Adrenal Androgen Testing

Assessment is indicated in patients with:

• Hirsutism
• Virilization
• Polycystic ovary syndrome (PCOS)
• Menstrual irregularities
• Infertility
• Precocious puberty
• Suspected adrenal tumors
• Congenital adrenal hyperplasia (CAH)

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DHEAS Measurement

Clinical Significance

• Serum DHEAS is the most commonly measured adrenal androgen because it is produced almost exclusively by the adrenal glands.

Elevated DHEAS Levels

May indicate:

• Congenital adrenal hyperplasia
• Adrenal adenoma
• Adrenal carcinoma
• ACTH excess
• Adrenal hyperplasia

Markedly elevated DHEAS levels are particularly suggestive of an adrenal neoplasm.

Reduced DHEAS Levels

May occur in:

• Adrenal insufficiency
• Hypopituitarism
• Aging
• Chronic glucocorticoid therapy

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Androstenedione Measurement

Androstenedione assessment helps evaluate androgen excess disorders.

Elevated Levels

Seen in:

• Congenital adrenal hyperplasia
• Polycystic ovary syndrome
• Adrenal tumors
• Ovarian androgen-producing tumors

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Adrenal Androgens in Congenital Adrenal Hyperplasia

Congenital Adrenal Hyperplasia (CAH) is a group of inherited disorders characterized by defects in steroid hormone synthesis.

The most common form is:

21-Hydroxylase Deficiency

Results in:

• Decreased cortisol production
• Increased ACTH secretion
• Adrenal hyperplasia
• Excess androgen production

Key Laboratory Findings

• Elevated DHEAS
• Elevated androstenedione
• Elevated 17-hydroxyprogesterone

Measurement of 17-hydroxyprogesterone is the primary screening test for CAH.

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Adrenal Androgens in Hirsutism and Virilization

Women presenting with excessive hair growth or masculinization should undergo adrenal androgen evaluation.

Elevated DHEAS

Suggests an adrenal source of androgen excess.

Common Causes

• Adrenal hyperplasia
• Adrenal adenoma
• Adrenal carcinoma
• ACTH-dependent disorders

Very high DHEAS concentrations warrant investigation for an adrenal tumor.

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Laboratory Methods

Adrenal androgens can be measured using:

• Immunoassays
• Chemiluminescent assays
• Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS)

LC-MS/MS provides superior specificity and accuracy for steroid hormone analysis.

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Clinical Interpretation

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Congenital Adrenal Hyperplasia Testing

Pathophysiology of CAH

The adrenal cortex synthesizes:

• Cortisol
• Aldosterone
• Adrenal androgens

Defects in steroidogenic enzymes block normal hormone synthesis.

As cortisol production decreases:

• ACTH secretion increases
• Adrenal cortex undergoes hyperplasia
• Steroid precursors accumulate
• Excess adrenal androgen production occurs

This results in varying degrees of:

• Virilization
• Salt-wasting
• Adrenal insufficiency

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Types of Congenital Adrenal Hyperplasia

• Several enzyme deficiencies can cause CAH.

1. 21-Hydroxylase Deficiency (90–95% of Cases)

• Most common form.

Features:

• Reduced cortisol production
• Reduced aldosterone production (severe forms)
• Increased androgen production

2. 11β-Hydroxylase Deficiency

Features:

• Reduced cortisol
• Increased adrenal androgens
• Hypertension due to excess deoxycorticosterone

3. 17α-Hydroxylase Deficiency

Features:

• Reduced cortisol
• Reduced sex steroid production
• Hypertension
• Sexual development abnormalities

4. 3β-Hydroxysteroid Dehydrogenase Deficiency

Rare disorder affecting synthesis of all adrenal steroids.

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Indications for CAH Testing

Laboratory testing is indicated in:

Neonates

• Ambiguous genitalia
• Salt-wasting crisis
• Failure to thrive
• Persistent vomiting
• Dehydration

Children

• Precocious puberty
• Accelerated growth
• Early development of pubic hair

Adolescents and Adults

• Hirsutism
• Menstrual irregularities
• Infertility
• Suspected non-classical CAH

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Initial Laboratory Evaluation

The diagnosis of CAH is based on hormonal assessment.

Key investigations include:

• 17-Hydroxyprogesterone (17-OHP)
• Cortisol
• ACTH
• DHEAS
• Androstenedione
• Testosterone
• Electrolytes

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17-Hydroxyprogesterone (17-OHP)

• Gold Standard Screening Test

Measurement of 17-hydroxyprogesterone is the primary laboratory test for diagnosing CAH due to 21-hydroxylase deficiency.

Physiological Basis

• 21-hydroxylase normally converts:
• 17-Hydroxyprogesterone → 11-Deoxycortisol

When the enzyme is deficient:

• 17-OHP accumulates
• Cortisol production decreases
• ACTH increases

Interpretation

Markedly Elevated Levels

Strongly suggest:

• Classical 21-hydroxylase deficiency

Mildly Elevated Levels

May indicate:

• Non-classical CAH
• Heterozygous carrier state

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ACTH Stimulation Test

The ACTH stimulation test is performed when basal 17-OHP levels are borderline or inconclusive.

Procedure

1. Measure baseline 17-OHP.
2. Administer synthetic ACTH.
3. Measure 17-OHP after 60 minutes.

Interpretation

Exaggerated elevation of 17-OHP following ACTH administration confirms:

• 21-hydroxylase deficiency

The ACTH stimulation test is particularly useful in diagnosing non-classical CAH.

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Serum Cortisol Measurement

Findings

Classical CAH

• Low cortisol concentration

Non-Classical CAH

• Normal or mildly reduced cortisol

Assessment of cortisol helps determine disease severity and adrenal reserve.

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Plasma ACTH Measurement

Due to reduced cortisol production:

• ACTH secretion increases through loss of negative feedback.

Findings

• Elevated ACTH levels in untreated CAH

ACTH levels are useful for monitoring treatment effectiveness.

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Electrolyte Assessment

• Electrolyte testing is essential in severe CAH.
• Salt-Wasting Form

Laboratory findings include:

• Hyponatremia
• Hyperkalemia
• Metabolic acidosis

These abnormalities result from aldosterone deficiency.

Clinical Importance

Untreated salt-wasting CAH may cause:

• Severe dehydration
• Hypovolemic shock
• Death in neonates

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Newborn Screening for CAH

• Many countries include CAH in routine newborn screening programs.
• Screening Marker

Dried blood spot measurement of:

• 17-Hydroxyprogesterone

Benefits

Early diagnosis allows:

• Prevention of adrenal crisis
• Early treatment
• Improved growth and development
• Reduction of morbidity and mortality

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Molecular Genetic Testing

• Genetic analysis can identify mutations in steroidogenic enzyme genes.

Common Gene

CYP21A2

Responsible for:

• 21-hydroxylase deficiency

Clinical Uses

• Diagnostic confirmation
• Carrier detection
• Family screening
• Prenatal counseling

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Imaging Studies

Imaging is not routinely required for diagnosis but may be useful in selected cases.

Adrenal Imaging

May reveal:

• Bilateral adrenal enlargement

Pelvic Ultrasound

Useful in:

• Evaluation of ambiguous genitalia
• Assessment of internal reproductive organs

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Monitoring Patients with CAH

Patients receiving glucocorticoid therapy require regular biochemical monitoring.

Follow-Up Tests

• 17-Hydroxyprogesterone
• ACTH
• Androstenedione
• Testosterone
• Electrolytes

Treatment Goals

• Adequate cortisol replacement
• Prevention of androgen excess
• Normal growth and development
• Avoidance of overtreatment

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Laboratory Findings in Common Forms of CAH

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Factors Affecting Adrenal Function Tests

Several variables can influence results.

Physiological Factors

• Stress
• Exercise
• Pregnancy
• Sleep patterns

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Medications

Increase Cortisol

• Oral contraceptives
• Estrogens

Affect Dexamethasone Test

• Phenytoin
• Carbamazepine
• Rifampicin

Affect Renin-Aldosterone System

• Diuretics
• ACE inhibitors
• Beta blockers

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Collection Factors

Improper timing of specimen collection can significantly alter results due to hormonal circadian rhythms.

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Interpretation of Adrenal Function Tests

Laboratory results should always be interpreted alongside:

• Clinical history
• Physical examination
• Imaging studies
• Medication history

A single abnormal hormone value rarely establishes a diagnosis.

Dynamic endocrine testing often provides more reliable diagnostic information than isolated hormone measurements.

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Clinical Disorders Diagnosed by Adrenal Function Tests