Steroid Hormones and Metabolism

Steroid Hormones

• These are steroids functioning as hormones.
• Secreted by adrenal cortex, testes, ovaries, and placenta during pregnancy.
• All are derived from cholesterol.
• Transported through the bloodstream to cells, regulating many physiological functions.

Steroid Hormone Metabolism

• The liver is the primary site.
• The kidney and gastrointestinal tract also perform metabolic transformations
• Biochemical steps neutralize biological activity and facilitate elimination:
  • Introduction of a hydroxyl group (e.g., estradiol to estriol).
  • Dehydrogenation (e.g., testosterone to androstenedione).
  • Reduction of double bond (e.g., cortisol to dihydrocortisol).
  • Conjugation of a hydroxyl group with glucuronic acid (e.g., testosterone to testosterone glucuronide).
• Conjugation with sulfuric or glucuronic acid is the most efficient metabolic process for excretion in the urine.
• Most steroid metabolites are excreted as water-soluble glucuronides or sulfates.

Steroid Hormone Chemical Structure

• Steroids contain a cyclopentanoperhydrophenanthrene nucleus.
• The phenanthrene nucleus consists of three six-sided rings (A, B, and C), with a D or cyclopentane ring attached.

Steroid Hormone Biochemistry

• Human steroid hormones are synthesized from cholesterol in the adrenal glands and gonads.
• Cholesterol comes from LDL cholesterol in the circulation.
• LDL uptake occurs via cell-surface LDL receptors on the adrenal gland, releasing cholesterol for steroidogenesis.
• The transport of intracellular cholesterol to sites of steroidogenesis is mediated by steroidogenic acute regulatory protein (StAR), regulated by adrenocorticotropic hormone (ACTH).
• The rate-limiting step means that without this step, the biochemical process will not occur

Classification

• Adrenocortical steroids are classified as corticosteroids and adrenal androgens.
• Corticosteroids include the mineralocorticoids and glucocorticoids.
• These steroids are the most physiologically important group of adrenal steroids.

Mineralocorticoids

• Mineralocorticoids help regulate salt homeostasis (sodium conservation and potassium loss) and extracellular fluid volume.
• Aldosterone: the most potent mineralocorticoid, synthesized in the zona glomerulosa of the adrenal cortex.
• Aldosterone synthase is an obligatory enzyme in adrenal cortex synthesis.
• Aldosterone is secreted at approximately 200 µg/day.
• Mineralocorticoids bind to a cytoplasmic mineralocorticoid receptor (MR) in the distal convoluted tubule, collecting duct of the nephron, colon, and salivary glands.
• They promote sodium reabsorption, potassium, and hydrogen ion excretion.

Glucocorticoids

• Cortisol is the major glucocorticoid, synthesized from cholesterol in the zona fasciculata and reticularis of the human adrenal cortex.
• It is secreted at approximately 25 mg/day.
• When released into circulation, cortisol mainly binds to corticosteroid-binding globulin (CBG).
• Over 95% of cortisol and cortisone conjugates with glucuronic acid and is excreted as a conjugate in urine.
• Less than 2% of cortisol is excreted unmetabolized as urinary free cortisol (UFC).
• Glucocorticoids bind to the glucocorticoid receptor (GR) in many tissues, including lymphocytes, hepatocytes, and bone.
• Glucocorticoids affect glucose and carbohydrate metabolism:
  • They increase synthesis of gluconeogenic enzymes like glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, increasing blood glucose.
  • They increase liver glycogen content through activation of glycogen synthase.
  • They inhibit glycogen phosphorylase, producing insulin resistance in muscle and adipose tissue, which further increases blood glucose concentrations.
• Cortisol increases catabolism of proteins in multiple tissues, with excess cortisol producing myopathy, thinning of skin, loss of strength in connective tissues, and bone loss which can lead to fractures.
• It affects lipid metabolism:
  • It redistributes adipose tissue centrally to the trunk, neck, and face (Cushing's Syndrome).
  • It increases adipocyte differentiation.
  • It promotes lipogenesis in these tissues.
  • It increases VLDL and triglyceride concentrations.
  • It decreases HDL and HDL-cholesterol concentrations.
  • It increases activity of adipose tissue hormone-sensitive lipase, allowing triglyceride breakdown and increased free fatty acid delivery to the liver.
• Cortisol are anti-inflammatory hormones that suppress:
  • The activity of pro-inflammatory enzymes (such as, cyclooxygenase 2 [COX-2]) and inducible nitric oxide synthase (INOS)
  • Various interleukins (IL-1, IL-2, and IL-6)
  • Tumor necrosis factor-alpha
  • Interferon-gamma
  • E-selectin
• At physiological concentrations, glucocorticoids help maintain vascular tone and cardiac output and stabilize lysosomal membranes.
• Glucocorticoids suppress hypersensitivity responses by inhibiting the production of histamine by basophils and mast cells.

Adrenal Androgens

• Adrenal androgens are Dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA-S), and Androstenedione.
• They provide androgenic effects through their peripheral conversion to testosterone, which binds to the androgen receptor (AR).
• "Sex hormones" gives secondary characteristics
• Important roles in reproductive health and body development.
• Without these adrenal androgens, there is no puberty.

Steroid Hormone Regulation

• Steroid hormones are not stored in hormone-producing cells and therefore must be produced as needed.
• Steroids are lipophilic which makes them pass through cell membranes to exit the hormone-producing cells, enter the circulation to be distributed throughout the body, and enter the cytoplasm of target cells.
• Translocation of the hormone-receptor complex to the nucleus initiates the action of the hormone.

Aldosterone Regulation

• Low blood pressure (hypovolemia), low blood volume, or low sodium levels result in the triggering of Renin-Angiotensin-Aldosterone System (RAAS).
• ↓ Blood pressure →↑ Renin (from juxtaglomerular cells in the kidney)
• Renin converts angiotensinogen → Angiotensin I
• ACE (from lungs) converts Angiotensin I → Angiotensin II
• Angiotensin II stimulates aldosterone secretion from the adrenal cortex
• Aldosterone increases sodium and water reabsorption in the kidneys → ↑ Blood volume & pressure

Serum Potassium Levels

• Hyperkalemia (↑ K+) directly stimulates aldosterone release.
• Aldosterone promotes K+ excretion in the distal tubules of the kidney.

ACTH (Adrenocorticotropic Hormone)

• Has a minor and transient effect on aldosterone secretion.
• More important for cortisol regulation.

Plasma Sodium Levels

• Indirect influence: Hyponatremia can activate RAAS.

Atrial Natriuretic Peptide (ANP)

• Released by the atria in response to↑ blood volume.
• Inhibits aldosterone secretion, acting as a counter-regulatory hormone.

Cortisol Regulation

• Stress or circadian rhythm stimulates the hypothalamus to release Corticotropin-Releasing Hormone (CRH).
• CRH acts on the anterior pituitary, stimulating the release of ACTH (Adrenocorticotropic Hormone).
• ACTH acts on the adrenal cortex (zona fasciculata), stimulating cortisol production and secretion.
• Cortisol then exerts negative feedback: on both the hypothalamus (↓ CRH) and anterior pituitary (↓ ACTH).

Circadian Rhythm Influence

• Cortisol levels follow a diurnal pattern:
  • Highest: Early morning (around 6-8 AM).
  • Lowest: Late evening/early night (around midnight).
• This rhythm can be altered by shift work, chronic stress, or disease.

Other Factors Influencing Cortisol

• Stress: Physical, emotional, illness, trauma → ↑ CRH and ACTH → ↑ Cortisol.
• Hypoglycemia: Low blood sugar is a potent stimulus.
• Inflammation/IL-6: Can stimulate HPA axis.

Adrenal Androgens Regulation

• Not well understood.
• The best characterized regulator of androstenedione and DHEA secretion, however, is ACTH.
• A diurnal rhythm in adrenal androgen concentrations parallels cortisol variations.
• Nevertheless, ACTH regulation of adrenal androgens does not explain the normal prepubertal and pubertal increases in adrenal androgen synthesis that occur in both boys and girls because ACTH does not increase prior to puberty.

Circulation of Steroid Hormones

• Steroid hormones (like cortisol, aldosterone, estrogen, testosterone) are lipid-soluble (lipophilic).
• Hydrophobic properties prevent them from freely dissolving in plasma and therefore require carrier proteins in the blood (90-98% are bound).
• Carrier proteins include:
  • Albumin (non-specific, binds weakly to many hormones)
  • CBG (Corticosteroid-Binding Globulin) – specific for cortisol and aldosterone
  • SHBG (Sex Hormone-Binding Globulin) – binds testosterone, estrogen
• Bound hormone = inactive, acts as a reservoir.
• Free hormone = active, can enter cells and bind to receptors.
• Only the free, unbound form crosses cell membranes, binds intracellular (nuclear) receptors, and exerts biological effects.
• Unbound steroids in the plasma are mostly conjugated to sulfate or glucuronic acid.

Steroid Hormone Metabolism

• The liver is the major site of steroid metabolism via P450 enzymes with the kidney playing less of a metabolic role but an important excretory role.
• Clearance of steroid hormones involves:
  • Hydroxylation
  • Dehydrogenation
  • Reduction of double bonds
  • Conjugation to sulfates or glucuronides
• The reduction in steroid concentrations increases their solubility and provides functional sites (such as, hydroxyl groups) or their conjugation to sulfate or glucuronic acid.
• Approximately 90% of conjugated steroids are excreted by the kidney.

Urinary Metabolites

• Measurement of the urinary excretion of adrenocortical hormones may be useful in the laboratory assessment of adrenal disease.
• However the difficulty of accurately collecting timed urine samples remains a challenge, and serum or plasma steroid assays are often preferred
• Immunoassays are available for the immunoassays of the major circulating steroid hormones are widely available

Dynamic Tests of Adrenal Function

• Designed to differentiate between primary and secondary causes of disease, as well as to detect abnormalities with baseline tests
• A stimulus is applied and the resultant release of a hormone is measured over a specific period.
• Suppression tests assess the integrity of feedback mechanisms
• ACTH stimulation test documents the functional capacity of the adrenal glands to synthesize cortisol.
• The adrenal glands are stimulated by cosyntropin (also called tetracosactrin; Synacthen and Cortrosyn are brand names).
• Cosyntropin is a synthetic polypeptide that is the N-terminal 24 amino acid sequence of ACTH and contains the biologically active domain of the hormone
• Important to avoid false positive results in ACTH stimulation tests
• The test determines whether the adrenal glands are responsive to ACTH

CRH Stimulation Test

• A direct and selective test of anterior pituitary gland function.
• Injection of ovine CRH stimulates ACTH secretion in healthy subjects within 60 to 180 minutes.
• Glucocorticoids inhibit this effect (as in cases of Cushing syndrome resulting from an adrenal adenoma or ectopic ACTH secretion by a tumor).

Insulin-Induced Hypoglycemia Stimulation Test

• In the insulin-induced hypoglycemia stimulation test, insulin is given to produce hypoglycemia which is a physiologic stimulus for release of CRH.
• To test the integrity of the hypothalamic-pituitary-adrenal axis indirect tests of ACTH secretion rely on the adrenal response to signals that stimulate ACTH release.
• Plasma ACTH or cortisol concentrations are then measured and will be increased if the hypothalamic-pituitary-adrenal axis is intact
• This test involves the risks of hypoglycemia such as obtundation/decreased mental capacity, seizure, coma, and death, thus it should only performed under carefully supervised conditions
• Venous access must be maintained during the procedure

Metyrapone Stimulation Test

• A less risky indirect test of hypothalamic-pituitary-adrenal axis function involves the administration of metyrapone.
• Metyrapone is an inhibitor of the 11 ẞ-hydroxylase enzyme (CYP11B1) that converts 11-desoxycortisol to cortisol.
• The decrease of cortisol is expected to allow an increase in ACTH secretion.
• Several protocols have been designed to directly or indirectly monitor the effect of metyrapone on ACTH secretion.

Dexamethasone Suppression Test

• The principle of this test is based on the feedback mechanism between cortisol, ACTH, and CRH.
• The integrity of this feedback mechanism is assessed by administering the potent glucocorticoid dexamethasone, and measuring serum or urine cortisol concentrations to evaluate the hypothalamic response.
• Dexamethasone is chosen for suppression testing, because it does not significantly cross-react in cortisol immunoassays.
• Patients with Cushing syndrome of any cause will fail to suppress their morning plasma cortisol concentration to less than 2 µg/dL in response to a 1mg dose of dexamethasone administered at 22:00.

Mineralocorticoid Stimulation Tests

• Procedures for stimulating the renin-angiotensin system are based on volume depletion maneuvers, such as sodium restriction, upright posture, or diuretic administration
• Another simple and convenient stimulation test consists of sodium restriction and upright posture.

Furosemide Stimulation Test

• The diuretic furosemide (40 to 80 mg) is administered to decrease plasma volume, followed by 4 hours of upright posture.
• This test does not require hospitalization or special diets, although it is recommended that the patient maintain a diet with a normal salt intake.
• Dietary sodium is restricted to less than 20 mmol/day or 3 to 5 days; urine is collected or creatinine and sodium measurements until equilibrium with the new diet is established
• Plasma renin activity (PRA) is measured after 2 hours of standing

Mineralocorticoid Suppression Tests

• Procedures use saline infusion, oral salt loading, or mineralocorticoid administration, each of which should suppress the secretion of aldosterone by the adrenal gland

Disorders of the Adrenal Cortex

• Conditions include Adrenal Insufficiency (Addison Disease), Hypoaldosteronism, Glucocorticoid Excess (Cushing Syndrome), Congenital Adrenal Hyperplasia
• Related conditions are functioning Adrenocortical Tumors, Non Functioning Adrenocortical Tumors, and Mineralocorticoid excess (Hyperaldosteronism).

Choice of Specimen

• Steroid hormones are measured in urine, blood, saliva, and hair. Clinical assessment of adrenal function, blood (plasma or serum) measurements are convenient, but conditions that cause the binding protein concentration to increase may produce clinically benign elevations in the total cortisol concentration
• Urinary concentrations are helpful in determining time-integrated hormone production and provide useful estimates of free hormone concentrations in blood if an appropriately timed urine specimen collection is available
• Saliva has been proposed as an alternative to urinary hormone assays to estimate free hormone concentrations in blood, the principal advantage being ease of collection of saliva
• Hair analysis may be useful for assessing adrenal hormone production over an extended period of time, but currently hair testing is not in widespread use

Urine Collection

• Urinary excretion of hormone, or its metabolites, provides an approximation of the amount secreted over the time the urine is collected, typically 24 hours.
• Thus, urinary hormone or metabolite measurements are useful when the hormone has a very short biological half -life and/or its secretion is pulsatile or varies in predictable cycles
• Timed urine collections suffer variability associated with factors, such as completeness of collection and impaired renal function

Blood Collection

• Circulating concentrations of steroids in the blood provide the most direct measure of hormone secretion.
• Provocative testing often require blood samples to study the rapid changes that occur in hormone concentrations
• Diurnal variations of hormones must be taken into account when taking blood samples
• Steroid hormone concentrations are nearly the same in serum and plasma

Saliva (Oral Fluid)

• Most steroids of clinical interest have been measured in saliva, and for some steroids, such as cortisol, estriol, and progesterone, salivary concentrations appear to correlate with the free hormone concentration
• Saliva ease of collection (especially in pediatric patients)
• Many saliva collection devices have been created for this purpose (i.e. cotton swabs, cotton ropes, and hydrocellulose microsponges)
• Cortisol is better measured in directly collected saliva specimens
• Salivary cortisol concentrations measured around midnight are useful

Analyitical Methods

• Immunometric, chromatographic, and combined chromatographic/mass-spectrometric methods are available or measuring steroid hormones

Measurement of Total Cortisol

• Immunoassays are the most commonly used methods for measuring cortisol in serum and urine, and are widely available on various automated immunoassay platforms
• Most cortisol immunoassays in routine use are heterogeneous, competitive-binding assays that require no initial extraction of steroids from the specimen
• Cortisol is displaced from CBG and other endogenous binding proteins by protein-binding agents, low pH, or heat treatment
• Most contemporary automated cortisol immunoassays use non isotopic labels
• The products generated from these assays are measured using photometric, fluorescent, or chemiluminescent labels and detectors
• Nonenzymatic approaches involve fluorescent and chemiluminescent labels, and these are the most common methods currently in use
• Homogeneous immunoassays or cortisol include the enzyme-multiplied immunoassay technique (EMIT) and the cloned enzyme donor immunoassay (CEDIA)
• Chromatographic methods for measuring cortisol include gas chromatography (GC), thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and methods coupled with mass spectrometry (GCMS, LCMS, LCMSMS)
• Liquid chromatography/tandem mass spectrometry (LCMSMS) is increasingly preferred due to its simplified sample preparation and ability to measure multiple corticosteroids, making it likely to become the standard method for corticosteroid analysis

Specimen collection and Storage

• Cortisol is measured in serum, heparinized plasma, or EDTA plasma
• In serum or plasma specimens, cortisol is stable or 7 days at room temperature or refrigerated, and it is stable or 3 months frozen at -20 °C
• Blood cortisol levels follow a diurnal pattern, peaking in the morning and dropping to less than half by evening
• Cortisol increases with stress, glucocorticoid therapy, pregnancy, depression, and hypoglycemia
• There are no significant differences between sexes or across ages
• Cortisol has a short half-life of about 60 minutes, causing rapid changes in blood levels
• In newborns, cortisol briefly spikes after birth, drops within 12-48 hours, then stabilizes within a week

Measurement of Free Cortisol

• Free cortisol in serum can be measured using ultrafiltration, equilibrium dialysis, or gel filtration, but these methods are complex and costly
• Alternatives include estimating free cortisol using albumin and CBG levels or measuring urinary and salivary cortisol
• Urinary cortisol has long been used to screen for Cushing's syndrome, though salivary cortisol is now preferred for initial testing
• Salivary cortisol reflects free blood cortisol and is easy to collect, with minimal protein interference
• Freezing and centrifugation help clear saliva of interfering protein
• Care must be taken to avoid oral bleeding, which can falsely elevate results

Measurement of Aldosterone

• Measuring aldosterone is technically difficult due to its very low concentration in blood, about a thousand times less than cortisol
• Immunoassays, including radioimmunoassays (RIA) and newer nonisotopic methods, are commonly used
• LC-MS/MS is also used for aldosterone measurement, offering high sensitivity and good agreement with RIA results

Measurement of 17-Hydroxyprogesterone

• The most common cause of congenital adrenal hyperplasia (CAH) is 21a-hydroxylase (CYP21) deficiency, which leads to a buildup of 17-hydroxyprogesterone (17-OHP 17-OHP is also elevated in 11β-hydroxylase (CYP11B1) deficiency, making 17-OHP measurement key for diagnosing common forms of CAH
• In 21a-hydroxylase deficiency, 17-OHP levels can be hundreds of times above normal
• Radioimmunoassays and other immunoassay methods using various labels (photometric, fluorescent, chemiluminescent) are used to measure 17-OHP in serum, plasma, saliva, and amniotic fluid

Measurement of 11-Desoxycortisol

• 11-desoxycortisol is measured in serum or plasma to diagnose 11β-hydroxylase (CYP11B1) deficiency or during the metyrapone stimulation test
• In normal adrenal function, metyrapone causes a 40- to 80-fold increase in 11-desoxycortisol, so highly sensitive methods aren't necessary
• Methods uses radioimmunoassays, some assays include extraction or chromatography steps Nonisotopic methods include enzyme immunoassays, fluorometric, and fluorescence polarization techniques.