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INTRODUCTION

Although the number of patients with Endocrine hypertension constitutes a small portion of individuals with hypertension, identifying the cause could offer a cure for these patients. Among the common causes of endocrine hypertension are;

1. Renal vascular hypertension (RVH)

2. Primary aldosteronism (PA)

3. Glucocorticoid remediable aldosteronism (GRA)

4. 11-hydroxylase deficiency of congenital adrenal hyperplasia

5. Apparent mineralocorticoid excess (AME) syndrome

Several clues may suggest the presence of endocrine hypertension :

• The onset of hypertension in young individuals (<25 yrs) or after age of 50 yrs
• Worsening of hypertension despite maximum drug treatment
• Unprovoked hypokalemia
• Symptoms and signs suggestive of endocrine diseases such as hyperthyroidism, hypothyroidism, acromegaly, Cushing's syndrome, and pheochromocytoma
• Presence of abdominal bruit

COLLECTION OF SPECIMENS FOR LABORATORY TESTS COMMONLY USED IN DIAGNOSIS

ALDOSTERONE
CPT CODE:82088
SPECIMEN: 1 Gold Top, 3.5 mL blood
MINIMUM VOLUME: 2 mL blood
SPECIAL INSTRUCTIONS: May submit heparinized plasma. Do not use EDTA plasma. Results vary with sodium excretion, electrolytic balance and posture (standing or recumbent).
REFERENCE RANGE: Adult Female and Adult Male: (Standing) 4-31 ng/dL; (Supine) 1-16 ng/dl. Aldosterone levels should be correlated to sodium intake as reflected by 24 hour urine sodium excretion.

PLASMA RENIN ACTIVITY
CPT CODE:84244
SPECIMEN: 2 Lavender Tops, 5 mL blood and concurrent urinary sodium excretion in mEq/24 hr
MINIMUM VOLUME: 2 Lavender Tops, 5 mL blood
SPECIAL INSTRUCTIONS: Collect and transport at room temperature. Centrifuge and freeze the plasma immediately.
REFERENCE RANGE: Interpretation of plasma renin activity results requires a concurrent 24 hour urinary sodium excretion level, and knowledge of the patient's posture and medications. A nomogram, sodium excretion vs renin activity is reported with verified results. Normal adults on a regular sodium diet (urinary sodium excretion approximately 150 mEq/24 hr) in an upright position and on no medications have plasma renin activity levels of 1.0 to 3.8 ng/mL/hr.

CATECHOLAMINES (PLASMA)
CPT CODE:82384
SYNONYMS: Norepinephrine and Epinephrine
TEST INCLUDES: Plasma Norepinephrine, Epinephrine and Dopamine
SPECIMEN: Green Top, 6 mL blood
MINIMUM VOLUME: 2.5 mL plasma
SPECIAL INSTRUCTIONS: A catheter is placed in the vein 30 min before drawing the sample. Draw samples as follows: 1) Patient should be lying down for 30 minutes before having the supine specimen drawn into a chilled green top vacutainer tube. 2) Patient should then be instructed to sit upright for 15 minutes with the catheter still in place. 3) Draw the upright specimen into a chilled green top vacutainer tube. 4) Label tubes supine or upright, place on wet ice. 5) Send to the laboratory STAT.
REFERENCE RANGE: See text

CATECHOLAMINES (URINE)
CPT CODE:82382
SYNONYMS: Norepinephrine, Epinephrine
TEST INCLUDES: Norepinephrine & Epinephrine
SPECIMEN: Urine, timed specimen collected in Hydrochloric Acid, preferred specimen is a complete 24 hour collection..
MINIMUM VOLUME: 10 mL
SPECIAL INSTRUCTIONS: Must be collected in Container which contains 15 mL 6 N Hydrochloric Acid..
REFERENCE RANGE:
Adult Range: NE <100 ug/24 hr, E <25 ug/24 hr; Pediatric Range NE <2.0 ug/kg/24 hr; E <0.5 ug/kg/24 hr 

PLASMA FREE METANEPHRINES
CPT code- 83835
Available from Quest Diagnostics ( www.questdiagnostics.com)
SAMPLE-5 ml of frozen EDTA (lavender top) plasma
SPECIAL INSTRUCTIONS-  Patient must refrain from using acetaminophen for 48 hours before testing. Patient must refrain from using caffeine, medications, and tobacco, and from drinking coffee, tea or alcoholic beverages, for at least 4 hours before testing.
SPECIMEN CONTAINER- Plastic screw top vial, transport frozen.
STABILITY- Refrigerated-30 days: frozen- 30 days.
METHOD-HPLC

Renal vascular hypertension (RVH)

Most patients with RVH or renal artery stenosis (RAS) are older than 50 yrs of age and have renal atherosclerotic disease. Younger patients usually have renal artery fibromuscular dysplasia.
The suggested work-up for RVH/RAS depends greatly on the degree of the clinical suspicion of the presence of this disease. In general a low index of suspicion does not require any work-up. In presence of a high degree of suspicion, renal arteriogram and measurement of renal vein renin would help confirm the diagnosis and localize the site of the stenotic renal blood vessel. Individuals with moderate suspicion of RVH/RAS need to undergo screening and confirmation testing. Measurement of plasma renin activity (PRA) is not specific for diagnosing RAS. In this disease, PRA levels may range from normal to high. The sensitivity and specificity (1) of various tests are outlined in the following table:

1. Captopril test is used as a screening test. PRA is measured before and 2 hrs after oral administration of captopril 25mg in seated position. Patients with RVH/RAS respond by increasing PRA to greater than 12 ng/ml/hr with absolute increase by greater than 10 ng/ml/hr. This test is less specific and sensitive than duplex ultrasound, CTA and MRA.
2. Duplex ultrasound is used as a screening test. However, it is less sensitive in obese patients and results are operator dependent.
3. CTA and gadolinium-enhanced MRA can be used as diagnostic tests. They have the highest diagnostic performance for the detection of RAS among non-invasive tests for RAS.
4. The gold standard for confirming the diagnosis of RAS is renal angiogram.

Primary Aldosteronism

Drugs that influence the renin-angiotensin-aldosterone system may disturb the evaluation of primary aldosteronism. Before conducting any investigations for diagnosing this disease, any drugs that may interfere with renin or aldosterone measurements (spironolactone, Ca++ channel blockers, ACE inhibitors, angiotensin receptor blockers, beta blockers) should be stopped at least two weeks in advance with the exception of spironolactone that has a longer half-life and should be discontinued for six weeks in advance. Alpha blockers and alpha-methyldopa appear not to cause a problem.

Screening tests for primary aldosteronism:

1. Serum potassium. This test is not specific for diagnosing primary aldosteronism since a.significant number of patients with this disorder have normal plasma potassium levels and about 20% of patients with essential hypertension may have hypokalemia.
2. Plasma aldosterone concentration (PAC). PAC is influenced by salt intake and its level should be correlated to the preceding day 24h urinary sodium excretion. Disproportionate elevation of PAC in relation to 24h urinary sodium excretion is usually seen in patients with primary aldosteronism. However, this test does not differentiate between primary and secondary aldosteronism.
3. Plasma renin activity (PRA). Similar to PAC, sodium intake influences PRA. Suppressed PRA, for the level of the preceding day 24h urinary sodium excretion,. is suggestive of primary aldosteronism. Suppressed PRA does not differentiate between primary aldosteronism and low renin essential hypertension. In addition, other diseases such as excess mineralocorticoids other than aldosterone, apparent mineralocorticoid excess, and Liddle syndrome are associated with suppression of PRA.
4. The aldosterone-to renin ratio is an easy, inexpensive, and rapid means of screening for primary aldosteronism. To enhance the predictability of this ratio, blood samples should be obtained after the patient has been in upright position for 2 hours. The best correlation between PRA and PAC is achieved with low sodium intake, while the patients is in upright position. A ratio greater than 30 is suggestive but not diagnostic for presence of primary aldosteronism. Combination of this ratio with plasma aldosterone level greater than 20ng/dl increases the sensitivity and specificity of this test to 90% and 91% respectively (2). There are situations where the ratio may produce false negative or false positive results. 20 to 25% of essential hypertension patients have suppressed PRA and confirmatory test should be done. Hypokalemia lowers plasma aldosterone levels and potassium repletion before testing would avoid this problem. Specific medications may interfere with measuring PRA or aldosterone. Spironolactone increases aldosterone and it should be held at least 6 weeks before measuring renin or aldosterone. Beta-blockers lower PRA and produce false positive results. Calcium channel blockers could lower aldosterone levels and produce false negative results. Similarly, angiotensin converting enzyme inhibitors (ACEI) and angiotensin receptor blockers (ARBs) could produce false negative results through increasing PRA. Alpha-blockers and a-methyldopa (Aldomet) when used for short time during the work up of primary aldosteronism appear not to affect renin or aldosterone. The lower limit of detection varies among different PRA assays and can have a dramatic effect on the PAC/PRA ratios (3).
   5. Post captopril PAC/PRA enhances the accuracy for diagnosing primary aldosteronism. A ratio greater than 35 has sensitivity and specificity of 100% and 67-91%, respectively, compared with 95.4% and 28.3% respectively at baseline in patients with primary aldosteronism (4). This test appears to be as sensitive as salt loading in confirming a diagnosis of primary aldosteronism (5). This test is done by administering 25 mg of captopril orally, taken 2 h before sampling (6).

Confirmatory tests for primary aldosteronism:

1. Measuring 24H urinary excretion of aldosterone after 3 days of high salt intake (>200meg/day). 24h urinary sodium and creatinine should be measured simultaneously to ensure the high sodium intake and the adequacy of urine collection. Failure of high salt to suppress urinary aldosterone excretion to <11µg/24h is diagnostic for primary aldosteronism. This test has a sensitivity of 96% and specificity of 93% for PA (7,8).
2. Fludrocortisone suppression test is done by giving fludrocortisone 0.1mg PO Q 6h or 0.2 mg PO Q12h and oral sodium chloride greater than 200 mmol PO per day for 4 days. Failure to suppress upright PAC to less than 5ng/100ml by day 4 confirms the diagnosis of primary aldosteronism. Upright PRA should be suppressed to less than 1ng/ml/h on day 4 of the test. Since hypokalemia inhibits aldosterone secretion, potassium chloride supplement should be given to keep plasma potassium levels close to the normal range. This test is considered the most sensitive test to diagnose primary aldosteronism.
3. Saline suppression test is done by measuring upright PAC before and again supine after intravenous administration of 500 ml/h of 0.9% sodium chloride for 4 hours. Failure to suppress PAC to less than 6ng/100ml at the end of this test confirms the diagnosis of primary aldosteronism (9). This test is easy to do on outpatient basis.
   Both fludrocortisone and saline suppression tests are contraindicated in patients with severe hypertension or congestive heart failure (5).

Differentiating between Aldosterone Producing Adenoma Vs. Bilateral Adrenal Hyperplasia (BAH)

1. Changes in PAC on upright posture. Patients with aldosterone producing adenoma (APA) show no change or reduction in PAC on upright posture. Patients with bilateral adrenal hyperplasia (BAH) show an increase in PAC on upright posture. This test is done by measuring PAC in supine position and after 4 hours of upright posture. 70% of patients with BAH respond by increasing PAC of at least 50%.
2. Computed tomography (CT) scanning and MRI have poor sensitivity in localizing small APA (<5mm in diameter). For APA greater than 5mm, CT scan is easier, faster and cheaper to perform and has sensitivity similar to MRI.
3. Bilateral adrenal venous sampling (AVS). This test differentiates APA from BAH and preoperatively lateralizes the side of APA. AVS should be done if CT scan is negative and after excluding glucocorticoid remediable aldosteronism. PAC and cortisol levels are measured in the inferior vena cava (IVC) and right and left renal arteries before and 30 to 60 minutes after intravenous injection of synthetic ACTH, cosyntropin 0.25 mg. The purpose of measuring plasma cortisol is to confirm the site of the sampling catheter. Plasma cortisol levels are much higher in adrenal veins than IVC. ACTH acutely stimulates aldosterone secretion and will help magnify the differences in PAC levels between the two adrenal glands in case of APA. The aldosterone/cortisol ratio (A/C) of the involved to contralateral side provides the best diagnostic accuracy for determining if one adrenal is responsible for increased aldosterone production. With determination of bilateral selective samples,  ratios of (A/C on involved side) / (A/C of IVC) > or = 1.1, or of (A/C involved side) / (A/C opposite  side) > or = 2 provide the best compromise of sensitivity and false positive rates for lateralization of the etiology of Primary Aldosteronism(9a).

Glucocorticoid Remediable Aldosteronism (GRA)

1. GRA should be suspected in patients with early onset of hypertension or presence of family history of mortality or morbidity from early hemorrhagic stroke.
2. PRA is suppressed unless patient was treated with spironolactone or amiloride. PRA level is non-specific since 20% of patients with essential hypertension have suppressed renin.
3. PAC or urinary aldosterone is normal or mildly elevated. PAC/PRA ratio is elevated > 30. Also, Plasma aldosterone fails to rise or falls during 2 h of upright posture following overnight recumbency (10,11,12).
4. Elevated urinary levels of 18-hydroxy-cortisol and 18-oxo-cortisol (18-oxo-F) (13,14). An elevated level of these compounds in 24-h urine collection is highly sensitive and specific for the diagnosis of GRA. Although 18-oxo-F is produced in aldosterone producing adenoma (APA), its level is 20-30 times higher in GRA than APA. The drawback of this test is that it requires 24-h urine collection.
5. The degree of hypertension, hypokalemia, urinary 18-oxo-cortisol, suppressed PRA or elevated plasma aldosterone can not be used to identify patients with GRA as they lack specificity, occurring also in other types of primary aldosteronism.
6. Aldosterone is suppressed to less than 4ng/dl (15) by dexamethasone 0.5 mg PO Q6h for 2-4 days (12,16,17), and is markedly elevated in response to ACTH administration (12,18). PAC post dexamethasone<4ng/dl confirms the diagnosis of GRA. Short dexamethasone suppression test (DST) may result in false positive results while long test could produce false negative results. Dexamethasone may also suppress aldosterone in patients with aldosterone producing adenoma (APA). However, since aldosterone secretion is autonomous, DST fails to suppress it to very low levels. Although DST is highly sensitive and specific for GRA (19,20), some patients may show initial suppression only to rise again by day 4 of treatment or fail to suppress PAC to < 4ng/dl) (19,20). The major drawback of DST is the need for multiple blood tests with either hospitalization or repeated outpatient visit. Also, it is difficult to perform this test in children.
7. Genetic testing using long PCR-based methods for detecting the hybrid GRA gene. This is a highly reliable test, with 100% sensitivity and specificity, and requires only a single blood sample for leukocyte or extracted DNA (21,22). This is a fast, cheap test and is as powerful as the southern blot test (23). This test can be used as screening test for neonates born to affected parents. Placental tissue or cord blood (2/1,22) could be used. A negative test eliminates the possibility of GRA diagnosis.

Pheochromocytoma

1. Measurements of plasma or urinary fractionated metanephrine and noremetanephrine by liquid chromatography coupled with mass spectrometry provide the most reliability and specificity (24) and avoid interferences by many drugs or diet. Determination of urinary catecholamines by positive-ion electrospray tandem mass spectrometry has proven to be fast (3.5 min instrumental run time) and free of interference from drugs and drug metabolites (25)
2. Since the concentration of normetanephrine sulfate and metanephrine-sulfate in plasma are about 20-30 fold higher than the levels of their free metabolites, the measurements of their deconjugated metabolites in plasma provides major advantages over their traditional measurements (26).
3. Plasma free metanephrines reflects direct production by the tumor tissue and is considered the best test for excluding or confirming pheochromocytoma. Free metanephrine production is continuous and independent of catecholamine release (27). Thus, measurement of plasma free metanephrines is more reflective of the tumor production than catecholamines and normetanephrine levels (normal 112pg/ml and 61 pg/ml respectively) measured by HPLC (greater than 400 pg/ml and 220 pg/ml respectively) strongly suggest presence of pheochromocytoma.
4. HPLC method should be used in the measurement of all catecholamines and its metabolites (28). Most patients with pheochromocytoma have elevation of one or more the following levels:
   • Urinary norepinephrine >30 µg/24h (normal 80µg/24h)
   • Urinary epinephrine >50 µg/24h (normal 20 µg/24h)
   • Urinary normetanephrine (normal 540 µg/24h for men an 310 µg/24h for women) >1400 µg/24h
   • Urinary metanephrine > 1000 µg/24h (normal 240 µg/24h for men and 140 µg/24h for women)
   • Urinary total metanephrines > 2mgl 24h (norma 1-2 mg/24h)
   • Urinary vanillylmandelic acid > 12mg/24h (normal 7-9 mg/24h)
   • Plasma norepinephrine > 2000 pg/ml (normal 498 pg/ml)
   • Plasma epinephrine > 400 pg/ml (normal 83 pg/ml)
   • Plasma metanephrines >220 pg/ml

Sensitivity and specificity of biochemical tests for diagnosis of pheochromocytoma (28,29)

1. Patients with hereditary pheochromocytoma should be tested at periodic intervals
   regardless of suspicious signs and symptoms.
2. Once the diagnosis of pheochromocytoma is confirmed, efforts should be directed toward the localization of the tumor.
3. Provocative catecholamine stimulation tests may provoke severe hypertension and should be avoided (30).
4. Clonidine suppression test: Plasma catecholamines should be measured before and after the administration of clonidine. Catecholamines should be suppressed with clonidine in patients with essential hypertension, but not in patients with pheochromocytoma (31,32).
5. Nuclear scans such as MlBG (sensitivity 88% and specificity 89%) help identify the site of catecholamine production in particular when CT (sensitivity 100% and specificity 50%) or MRI imaging fail to locate the lesion or their findings is inconsistent with biochemical evaluation.

Congenital Adrenal Hyperplasia: 11ß-hydroxylase deficiency

1. Elevated serum levels of deoxycorticosterone (DOC), the principal steroid index of the 11ß-hydroxylase deficiency.
2. Elevated serum levels of 11-deoxycortisol (compounds)
3. Potassium depletion, however, this is variable
4. Suppressed renin production. The degree of hyporeninemia may vary widely (33)
5. Low plasma cortisol
6. Chronic elevation of ACTH
7. Elevated serum level of 17-hydroxy progesterone (17-OHP) and androstenedione and urinary pregnanetriol. The elevation in serum 17-OHP and urinary pregnanetriol is not as great as in 21-hydroxylase deficiency CAH.
8. Normalization of DOC is an indicator of adequate glucocorticoid therapy.

Apparent mineralocorticoid excess

1. Suppressed PRA, very low aldosterone
2. Severe hypokalemia
3. Metabolic alkalosis
4. The biochemical diagnosis can be made by measuring the ratio of cortisol to cortisone or the ratio of their metabolites, hydrocortisol to hydrocortisone
5. A more definitive diagnosis is established by measuring the level of tritiated water in plasma samples when 11-tritiated cortisol is injected. Tritiated water production is not measurable in patients with AME syndrome, while in normal subjects and heterozygotes, 65-80% of the tritiated label appears as tritiated water (33).