Tamara Spaic, R4

1. Adrenal cortex and HPA axis

2. Testing in Cushing’s syndrome

3. Review of the Endocrine Society Clinical Practice Guidelines (JCEM, May 2008)

4. Assessment of adrenal medullary function and disorders

5. Which test is the best?

4 g weight 2x5x1 cm Posteromedial surface of the kidney Retroperitoneal

Unenhanced CT PET scan

zona glomerulosa lacks

Only zona glomerulosa

Trophic hormone of zonae fasciculata and reticularis

Major regulator of adrenal androgen and cortisol production

Regulated by CRH (whose action is also potentiated by AVP -arginine vasopressin, and β adrenergic catecholamines)

Rapid synthesis and secretion of steroids (hormone levels rise within minutes)

POMC (proopiomelanocortin) - precursor

ACTH circadian rhythm generated in suprachiasmatic nc (signals CRH release)

Secreted with both circadian periodicity and ultradian pulsatility

Periodic secretory bursts at frequency of ~ 40 pulses/day Entrained by visual cues, light –dark cycle Continuous CRH administration desensitizes the ACTH

response Prolonged pulsatile CRH administration restores cortisol

secretion Corticosteroids directly suppress basal or stimulated

ACTH pulse amplitude

1. Episodic secretion and circadian rhythm of ACTH

2. Stress responsivness of the HPA

3. Feedback inhibition by cortisol of ACTH secretion

Physical stresses (major illness, surgery, trauma, starvation)

Psychological stress (anxiety, depression, manic-depressive psychosis)

CNS and pituitary d/s Cushing’s syndrome Liver d/s CRF Alcoholism

ACTH and cortisol secreted within minutes (Sx, hypoglycemia)

Abolish circadian periodicity (prolonged stress)

Stress →↑CHR → ↑ACTH Stress response abolished by proior high

dose glucocorticoids/Cushing’s syndrome Enhanced following adrenalectomy

FAST feedback inhibition – depends on the rate of increase of GC (not the dose)

Rapid (within minutes) and transient (up to 10 minutes)

DELAYED feedback inhibition is both time and dose dependent

Ideally nonstressed resting subject should have venous sample drawn 6-9 am

Unstable on room temperature (must immediately go on ice, -20° C)

Siliconized glass tube containing EDTARandom ACTH values do not on their own provide an accurate assessment of HPA function

Under basal (non stressed) conditions 8-25 mg/day (22-69 μmol/day) Mean 9.2 mg/day (25 μmol/day) – less than

thought before (?20 mg/day)

Circulate bound to plasma proteins (bound – biologically inactive

t½ (60-90 minutes) is determined

extend of plasma binding and

rate of metabolic inactivation 10% - free 75-90% - CBG (corticosteriod binding globulin) 15% - albumin (dexamethasone -75%)

Produced by the liver Progesterone in late pregnancy Synthetic steroids – do not significantly bind

(except prednisolone)

High estrogen states (pregnancy, OCP) ↑T4 Diabetes Hematologic d/s Chronic active hepatitis

Familial CBG deficiency ↓ T4 Protein deficiency states (nephrotic

syndrome, liver failure)

Metabolism – liver (conjugation) Excretion – kidney (<1% in urine unchanged

– “free”) 11β-hydroxysteriod dehydrogenase (inhibited

by IGF-1)

↓ clearance in starvation/anorexia nervosa and pregnancy (↑CBG)

↓ metabolism and excretion – hypoT4 ↑metabolism – OCP, liver disease Also drugs : dilantin, barbiturates, mitotane,

rifampin

ACTH Dependent ACTH Independent Pseudo-Cushing’s Syndrome

Pseudo-Cushing’s SyndromeEtohObesityDepression

Sign/symptom Frequency (%) Truncal obesity 96 Facial fullness 82 Diabetes or glucose intolerance 80 Gonadal dysfunction 74 Hirsutism, acne 72 Hypertension 68 Muscle weakness 64 Skin atrophy and bruising 62 Mood disorders 58 Osteoporosis 38 Oedema 18 Polydipsia, polyuria 10 Fungal infections 6

The mean age of the 239 female and 63 male patients was 38·4 years (SD 13·5; range 8–75).

• Answer 2 questions:

1. Does this patient have Cushing’s Syndrome?

2. Having confirmed Cushing’s syndrome clinically and biochemically – What is the cause?

• Diagnosis

1. UFC

2. Circadian Rhythm of plasma cortisol

3. Low dose dexamethasone

4. Salivary cortisol

• Differential Diagnosis

1. Plasma ACTH

2. Plasma K, HCO3

3. High dose dexamathasone suppression

4. Metyrapone test

5. CRH

6. IPSS

7. CT/MRI pituitary, adrenals

8. Scintigraphy

Obesity epidemics Aging population Suspicion - common Rare diagnosis (5-10 cases/million

population/year) – Cushing’s disease – (70% of all cases)

0.5% of lung cancer patients have ectopic ACTH syndrome

False positive tests

2-5% prevalence of unsuspected Cushing’s syndrome in pts with poorly controlled DM

0.5-1% in HTN 10.8% of pts with osteoporosis/vertebral # 3% in osteoporosis Overlap with PCOS (5.8%) 9% pts with incidental adreanal nodules (>2

cm) have evidence of hypercortisolism

• To reduce FP rate (high pretest probability)1. Pts with unusual features for age

(osteoporosis, HTN)2. Pts with multiple and progressive features

9especially those predictive of Cushing’s3. Children with decreasing height ‰ and

increasing wt4. Adrenal incidentaloma compatible with

adenoma

<1% of secreted cortisol unchanged in urine Cushing’s – CBG binding capacity exceeded, so plasma

free cortisol ↑ - ↑UFC N range 50-250 nmol/24 h (80-120) Not affected by conditions and meds that alter CBG Use the upper limit of normal as cut-off point Should do 2 or 3 complete consecutive collections (with 24

hr Cr) Can not do in RF (falsely low) False + if fluid intake >5 L /day

Am cortisol often not elevated in Cushing’s syndrome

(late night usually increased) Small increases in cortisol at circadian nadir may

not be detected as ↑ UFC Sn 45-71% (although most studies show

excellent sn, sp is the problem) Pseudo vs Cushing’s – ? Useful (also anxiety,

starvation, AN) To avoid overlap 4-fold increase Levels elevated during stress

Abnormal circadian rhythm (absence of late night nadir) Same as in midnight serum cortisol (but impractical) Sn 100%, Sp 77% CBG absent in plasma (measures free, not dependent on

CBG) Not affected on saliva amount or composition Stable on room temperature Can be sampled at home by the pt At least 2 measurements

N (bedtime or 2300-2400) <4 nmol/L Sn 92-100% Sp 93-100% (highly accurate for differentiating from

pseudocushing’s) Circadian rhythm is blunted in depression, shift workers May be absent in critically ill Chewing tobacco or licorice may have falsely elevated

result (inhibits which enzyme?) ?Smokers Different time zones

11 β hydroxysteriod dehydrogenase type 2

Cortisol → cortisone (inhibits)

Assessing feedback inhibition of HPA axis Suppresses pituitary ACTH ↓plasma and urinary cortisol Cushing’s – fails to suppress Dexamethasone does not interfere with the

measurment of cortisol Measure simultaneously daxamethasone

level (to assess compliance, etc)

1 mg po dexamethasone at 23 00 Am cortisol If cortisol <50 nmol/L – excluded (Sn 95%) FP 12.5% (dilantin, rifampin, chronically ill, etoh, uremia,

estrogen, pseudocushing’s) FN <2% (slow dex metabolizers)

Plasma cortisol at day 0 and 48 hours (9:00 am)

Dexamethasone 0.5 mg q6h x 48 hours FP <1% TP 97-100% (increased specificity) 2 weeks absence from etoh and 6 weeks of

OCP ?Pseudocushing’s r/o

Cushing’s syndrome (CS) - may adequately suppress serum cortisol (sn 98% if 50 nmol/L used) (? Impaired clerance)

Pseudocushings (PC) - ↑CRH secretion, yet cortisol continues to exert negative feedback on HPA (allowing suppression by exogenous GC)

CS – HPA axis more responsive to CRH and less to dex

CRH test (CS vs PCS)

O.5 mg dex q6h for 48 hrs In standard LDDST 6hrs post serum cortisol CRH – 2 hrs after the last dose – serum

cortisol, than iv 100 μg human rCRH (bw before and 15 min after)

LDDST – cut off 50 nmol/L LDDST CRH - <38 nmol/L (excluded CS)

Addition of CRH to LDDST does not improve the diagnostic accuracy. By adding CRH can not improve Sn beyond 100%, while sp went down to 67%

Pregnancy : UFC Epilepsy : UFC or late night salivary RF: 1 mg DST Cyclic Cushing’s syndrome : UFC Adrenal incidentaloma : 1 mg DST

1. Plasma ACTH

2. High dose dexamethasone suppression test

3. CRH

4. IPSS

ACTH dependent vs independent Adrenal tumors < 1 pmol/L CS : N (inappropriately) or elevated Problem in differentiating CD and Ectopic

ACTH syndrome (30% overlap) Higer in EAS ( >20 pmol/L ) Normal range 2-11 pmol/L

Rational : resetting of the negative feedback control to ACTH at higher level in CD

DDx of Cushings Disease vs Ectopic ACTH syndrome

Originally described by Liddle in 1960 2 mg q6h x 48 hours Original to demonstrate >50% drop in urinary 17

OH CS Liddle used only for adrenal dependent vs pituitary

(at that time EAS not even described

Plasma and urinary free cortisol (0 and 48 hrs)

>50% suppression Problem : 20-30% EAS will suppress ~20 – 30 % of CD will not suppress But 90% of pts will have CD Diagnostic accuracy of test only 76%

You want very specific test (to identified rare/few cases of EAS)

Improve sp by changing cut off 100% sp with suppression of >90% of UFC

Sn 81% Sp 66.7% There was no cut off point that would yield

100% specificity Diagnostic accuracy only

70%

Gold standard Function

1. Confirmation of pituitary ACTH secreting tumor

2. Localization

Simultaneous IPS and peripheral ACTH measuring

Before and after CRH 5 time points (0,2,3,5,10 minutes) Each petrosal sinus and peripheral vein IPS/peripheral ACTH > 2 → CD Absent gradient – EAS Sn 97%, sp 100% (if ratio pf 3 used)

Technically demanding!!! Proficient center Complications : referred auricular pain,

thrombosis, hemorrhage Central location

MRI CT

Specialized part of autonomic nervous system Sympathoadrenal system System of “fight or flight” Actions best characterized by the appearance of patient

in shock

Chromaffin cells (endocrine cells of adrenal medulla)- contain granules important in storage and secretion of catecholamines

In humans 85% of catecholamine store is epinephrine

Catecholamines : dopamine, norepinephrine, and epinephrine

Chromogranin A (CgA) – peptide stored and released with catecholamines by exocytosis (higher in HTN patients)

Catestatin – fragment of prohormone that inhibits further catecholamine release (antagonist at neuronal cholinergic receptor), low level may increase the risk of EHTN

Abbreviations: TH, tyrosine hydroxylase; AAD, amino acid decarboxylase; DβH, dopamine β-hydroxylase; PNMT, phenylethanolamine-N-methyltransferase.

Tyrosine – derived from ingested food or synthesized from phenylalanine in the liver

First step – rate limiting TH (expressed only in tissues that synthesize CCH) inhibited by

dopa, dopamine, NE, ?alpha-methyltyrosine (Rx of pheo) AADC – found in non-neuronal tissues (liver, kidney), methyldopa is

competitive inhibitor Dopamine taken up into chromaffin garnules DHB – only in vesicles of cells synthesizing CCH In the adrenal medulla NE returns to cytosol to be methylated by

phenylethanolamine-N-methyl transferase (PNMT) to form epinephrine PNMT – nonspecific (lung, kidney, pancreas, RBC), inducible by high

dose corticosteroids, ?angiotensin II

Stored in the chromaffin granules with several neuropeptides (neuropeptide Y, substance P, VIP, chromogranins, ACTH)

Released by exocytosis Response to many stressful stimuli (pain,

exercise, hemorrhage, anesthesia, hypoglycemia, anoxia..)

Secretion mediated by release of Acetylcholine from the terminals of preganglionic fibers

In circulation bound to albumin mostly

Once NE released in the synapse : 1. Reacts with α1 postsynaptic receptor 2. Reacts with presynaptic α2 receptor (down

regulates its own synthesis and release)3. Taken up into the cell by “uptake 1” (blocked

by TCA, cocaine) – main mode of removal4. Diffuse out and undergo degradationNote: NE can also be removed by extraneuronal

uptake by “uptake 2” (inhibited by corticosteroids)

MOA – regulates the CCH content of neurons, levels ↑progesteron, ↓estrogen

Peripheral circulating NE metabolized largely by COMT (catechol-O-methyltransferase)

COMT – found in most tissues (blood cells, liver, kidney, vascular smooth muscle)

Conjugation with sulfate or glucuronide

Rare and often unrecognized adrenal medulla tumor

Derived from chromaffin cells (paraganglioma if from extra-adrenal chromaffin cells: 10-15%)

Wide range of clinical presentation Associated with different familial disorders

(vHL, MEN2a, 2b, NF-1)

COMMON (>33% OF PATIENTS)LESS COMMON (<33% OF PATIENTS)

Hypertension (probably >90%)      Paroxysmal (50%)      Sustained (30%)      Paroxysms superimposed (about 50%)   Hypotension, orthostatic (10%-50%)   Headache (40%-80%)   Sweating (40%-70%)   Palpitations and tachycardia (45%-70%)   Pallor (40%-45%)   Anxiety and nervousness (20%-40%)   Nausea and vomiting (20%-40%)   Funduscopic changes (50%-70%)   Weight loss (60%-80%)

   Tremor   Abdominal pain   Chest pain   Polydipsia, polyuria   Constipation   Acrocyanosis, cold extremities   Flushing   Dyspnea   Dizziness, syncope   Convulsions   Bradycardia   Fever

Paroxysm (or pheo crisis) is the consequence of CCH release

CCH synthesis at increased rate likely due to lack of feedback inhibition of tyrosine hydroxylase

CCH produced in quantities that exceed the vesicular storage capacity – accumulate in cytoplasm – diffuse into the circulation

Most contain more NE than Epinephrine (unlike normal medulla)

May secrete other peptides

Not innervated (unlike adrenal medulla) so catecholamine release is not initiated by neural impulses

Precipitated by any movement that displaces abdominal content, vigorous palpation of abdomen, spontaneous hemorrhage within tumor, surgical manipulation

Drugs – opioids (fentanyl), amphetamines, decongestants, histamine, TCA, dopamine antagonists (metoclopromide), glucagon, ACTH, intraarterial radiographic contrast

24 hour urine catecholamines and metanephrines

Fractionated plasma free metanephrines Other tests (clonidine suppression test,

chromogranin A) No role (plasma catecholamines, 24- hour

urinary VMA, histamine, glucagon, tyramine stim tests)

Foods: coffee/decaffeinated drinks – inhibits adenosine – inhibits release of CCH (false elevated results); bananas – tyrosine, peppers (may cause confounding peaks on HPLC)

Drugs – long list Conditions: ALS, carcinoid, eclampsia, exercise,

GBS, hypoglycemia, Pb poisoning, AMI, pain, porphyria, acute psychosis, RF (decreases excretion)

Tricyclic antidepressants Levodopa Drugs containing adrenergic receptor agonists (eg, decongestants) Amphetamines Buspirone and most psychoactive agents Prochlorperazine Reserpine Withdrawal from clonidine and other drugs Ethanol Acetaminophen (may increase measured levels of fractionated

plasma metanephrines in some assays) Captopril (may cause confounding peak) Codeine

Fractionated metanephrines (normetanephrine, metanephrine), total metanephrines, urinary vanillylmandelic acid (VMA)

Should include urinary Cr to verify adequate collection

Ability to follow instructions and cost Should not be on TCA Diagnostic cutoffs based on normotensive

volunteers – may result in excessive FALSE POSITIVE testing

Plasma free normetanephrine and metanephrine

Requires overnight fast and intravenous cannula

Patient should be supine at least 20 minutes before collection

No tylenol for 5 days prior and avoid caffeinated beverages overnight

As CCH are metabolized within tumor cells, plasma levels of free metanephrines are very sensitive

Centrally acting α2 adrenergic agonist Suppresses the release of CCH from neurons but it does not

affect secretion for pheochromocytoma Confirmatory test – distinguish between pheo and false positive

increase in CCH (when ↑CCH but not diagnostic) Administered orally (0.3 mg) Plasma catecholamines or metanephrines measured before and

3 hrs post dose In essential HTN – plasma CCH and normetanephrine

concentrations decrease, while in pheo – remain increased Patient should not take diuretics (euvolemic), β-blockers, TCA

Released from the secretory granules Increased in 80% of patients with pheo Have circardian rhythm (lowest at 8:00 am,

highest 11:00 pm) Also secreted from extra-adrenal sympathetic

nerves Renal clearance Sensitivity 98% and Specificity 97% PPV 97% and NPV 98% (sporadic)Herbomez et al. An analysis of biochemical diagnosis of 66 Pheochromocytomas. European Journal of Endocrinology 2007,

156:569-75

Ideal test has 100% sensitivity and specificity Sensitivity =TP/TP+FN (negative test r/o diagnosis) Specificity =TN/TN+FP (positive test r/i diagnosis) PPV = TP/ TP+FP NPV = TN/TN+FN

For any biochemical test - Sn low and Sp high for hereditary (high suspicion) vs sporadic case

If the disease has low prevalence - ↑false positive

Multicenter cohort study, over 5 years Objective – to determine the biochemical test or

combination of tests that provide the best method of diagnosing pheochromocytoma

1003 pt tested, 858 included (85%) 443 sporadic, 415 hereditary Conformation of pheo dx required +path examination

of surgical resection, biopsy, or inoperable malignant pheochromocytoma based on imaging.

Excluded if lack of tumor in CT/MRI, - path examination of Sx or bx, and lack of disease on 2 years patient follow up

Since measurements of urinary fractionated metanephrines and plasma free metanephrines offer similar high sensitivity, a negative result for either test is equally effective for excluding pheo.

However, because urinary fractionated metanephrines have low specificity, test of plasma free metanephrines exclude pheo in many more patients without tumor.

Plasma free MN provide the best test for excluding or confirming pheo and should be the test of choice for diagnosis.

Single center, retrospective study, over 1 year

349 patients, 8(?) hereditary Objective as above

Results:

+LR 6.3 (plasma free MN) + LR 58.9 (24 hrs urine total MN and CCH) If prevalence of pheo is- 0.5% (screened HTN pt) –post test probability:

3% vs 23% - 5.1% (incidentaloma) : 25% vs 76%- 42 % (MEN2a) : 82% vs 98% In addition specificity of plasma free MN falls to

77% in pt above age 60

Suggested that measurement of fractionated plasma metanephrines may be the biochemical test of choice in high-risk patients (those with a familial syndrome or vascular adrenal mass).

However, in the more common clinical setting when sporadic pheochromocytoma is sought, particularly older hypertensive patients, measurement of 24-h urinary metanephrines and catecholamines may provide adequate sensitivity, with a lower rate of false positive tests.

Included 3 studies 56 pt with pheo and 445 without pheo Sensitivity 97-100% Specificities 82-100% Pooled +LR 5.77, -LR 0.02 (sporadic cases) Post-test probabilities : 2.8%, 23.7% and 0.01%,

0.11% Conclusion : useful to r/o pheo, but a positive

test only slightly increases suspicion when screening for sporadic pheochromocytoma.