Adrenal Flashcards
Differential diagnosis of secondary hyperaldosteronism (increased renin with increased aldosterone)
Physiological:
1. Pregnancy
2. Dehydration
Pathological
1. High BP
a) Intrinsic renal disease
b) Renin secreting tumour
c) Renovascular hypertension
2. Low/normal BP
a) Bartter syndrome
b) Gitelman syndrome
c) heart failure
d) cirrhosis
Pharmacological
1. diuretics
2. vasodilators
Preanalytical
1. Cryoactivation of renin with high aldosterone
DDx increased aldo:renin ratio/primary aldosteronism
Aldosterone-producing adenoma
Idiopathic adrenal hyperplasia (usually bilateral)
Glucocorticoid-remediable hyperaldosteronism
Rarely, unilateral adrenal hyperplasia
DDx hypokalaemic, alkalotic hypertension
Primary aldosteronism and the DDx thereof
Cushing syndrome
Cortisol resistance (familial/primary glucocorticoid resistance)
DOComa
17 hydroxylase deficiency (Liddle syndrome)
Apparent mineralocorticoid excess (pseudohyperaldosteronism)
What enzyme is responsible for conversion of cortisol to cortisone?
11beta hydroxysteroid dehydrogenase 2
Pathophysiology of apparent mineralocorticoid excess
Normally, cortisol exists in thousandfold concentrations compared to aldosterone, and would activate the mineralocorticoid receptor except for the fact that tissues sensitive to aldosterone produce 11beta hydroxysteroid dehydrogenase 2. This enzyme converts cortisol to cortisone, which does not activate the MR.
In HSD2 deficiency, cortisol is not deactivated to cortisone and activates the MR, resulting in hypokalaemic, alkalotic hypertension, however, aldosterone and renin levels are suppressed.
Pathophysiology of glucocorticoid remediable hyperaldosteronism
Chimeric gene duplication involving CYP11B1 and CYP11B2. The ACTH-responsive promoter region of CYP11B1 fuses with CYP11B2 aldosterone synthase. Results in ACTH-responsive aldosterone synthase production in the zona fasciculata. Suppression of aldosterone production by dexamethasone supports the diagnosis of GRA. Treatment involves suppression of aldosterone production by exogenous glucocorticoids.
Utility of 24 hour urine excretion
Assessing completeness of urine collection (70kg male normal range between 9.1 and 14.5 mmol/day). Correcting for different volumes of urine collection between patients.
What is 17OHP and where is it produced?
An endogenous progestogen produced in the Zona fasciculata (also: corpus luteum, gonads and placenta)
How is 17OHP produced
By the action of 3beta hydroxysteroid dehydrogenase and 17 hydroxylase from 17OH pregnenalone and progesterone respectively
Causes of false positive and false negative 17OHP
Positive: early luteal phase, premature neonates
Negative: unstimulated late afternoon sample, corticosteroid therapy
Indications for measurement of 17-OHP
Screening for non-classical CAH: baseline morning 17-OHP > 6nmol/L.
Diagnosis of non-classical CAH due to 21alpha-hydroxylase deficiency when baseline morning 17-OHP > 6nmol/L, as part of SST.
Synacthen simulation tests may be falsely normal with acute secondary hypocortisolism. What alternative tests can be done in this situation?
Insulin tolerance test
Overnight metyrapone test
Contraindications to saline suppression test
Uncontrolled hypertension
Hypokalaemia
Arrhythmia
Severe CCF
Renal failure
Types of CAH
- STAR
- Cholesterol side chain cleavage (CYP11A1)
- 3 beta hydroxysteroid dehydrogenase type 2 (HSD3B2)
- CYP17A1 mutations affecting 17 alpha hydroxylase and 17,20 lyase
- POR - cytochrome P450 oxidoreductase
- 11 beta hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2)
- 21 alpha hydroxlase deficiency (CYP21A2)
Most common newborn screening assay for 21alpha hydroxylase deficiency CAH?
DELFIA - time-resolved dissociation enhanced lanthanum fluorescence immunoassay
What are metanephrines?
Metabolites of catecholamines. Biomarkers of PPGL and neuroblastoma
Why are parasympathetic paragangliomas non-functioning?
They don’t produce catecholamines
Types of paraganglioma
Sympathetic (along sympathetic chain)
Parasympathetic (head and neck)
What are neuroblastomas?
Tumours, the origin of which we believe are neural crest cells (precursors of chromaffin and sympathetic neurons)
Predominant population presenting with neuroblastoma?
Children
Locations that neuroblastomas form (in order of most to least common)
adrenal gland
abdomen
thorax
neck/pelvis
Cause of hypertension in phaeo
Noradrenaline
Investigation for PPGL
- Biochemical (metanephrines - catecholamines not recommended)
- Imaging (CT/MRI initially, DOTATATE/FDG/MIBG for mets)
- Definitive diagnosis post-surgical resection of tumour (biopsy not recommended)
- Genetic testing
What percent of PPGL is associated with germline mutations?
40%
What are the 3 catecholamines?
Adrenaline
Noradrenaline
Dopamine
What are the 3 metanephrines?
Metanephrine
Normetanephrine
3-methoxytyramine
Name the enzyme responsible for conversion of noradrenaline to adrenaline
PNMT - Phenylethanolamine-N-methyl transferase
What is PNMT? Where is it found?
The enzyme responsible for conversion of norad to adrenaline. Expressed only in adrenal medulla but not sympathetic neurons under inducement by cortisol from adjacent adrenal cortex
Where does the majority of circulating noradrenaline come from?
Spillover from synapse of pre- and post-ganglionic sympathetic neurons
How are noradrenaline and adrenaline metabolised?
- Monoamine oxidase pathway (neuronal tissue)
- Catechol-O-methyl transferase pathway (only expressed in adrenal and GI tissue)
Which pathway of catecholamine metabolism is responsible for production of metanephrines?
COMT pathway
Where do the majority of circulating metanephrines come from and why?
Adrenal gland is responsible for 95% of circulating metanephrine and 25% circulating normetanephrine. Absence of COMT in sympathetic neurons means noradrenaline is metabolised to DHPG, not normetanephrine
Where do the majority of circulating metanephrines come from and why?
Adrenal gland is responsible for 95% of circulating metanephrine and 25% circulating normetanephrine. Absence of COMT in sympathetic neurons means noradrenaline is metabolised to DHPG, not normetanephrine
Advantages of metanephrines over catecholamines as a biochemical marker?
Metanephrines are less affected by surges of catecholamine release in response to sympathetic activity
Production of metanephrines is constant
Metanephrines more specific for adrenal gland as they are not produced by neurons
Main sources of dopamine?
GI tract and mesenteric organs and diet.
Minor sources of dopamine?
Kidney
Sympathetic neurons
Difference between total, fractionated and free metanephrines?
Metanephrines can be conjugated with sulfate and circulate in plasma in these sulfated forms. Free metanephrines refer to unconjugated single fractions. Total refers to both free and conjugated forms. Fractionated just means there is differentiation between metanephrine, normetanephrine and 3-MT, but is measuring total not free MN/NMN/3-MT
Preparation required to measure “total” metanephrines?
Need to de-conjugate sulphates by acidifying/extracting/boiling the sample. May be called “total”, “conjugated” or “de-conjugated” metanephrines.
How are free metanephrines measured?
Measurement without acid hydrolysis, AKA “unconjugated” metanephrines
In what clinical context does plasma free metanephrines testing perform best?
High pretest prevalence of PPGL - has a much higher sensitivity than urine tests. But you can’t really go wrong if you order plasma free mets.
Endocrine society recommendations on methods to be used for metanephrine determination
Liquid chromatography with MS or electrochemical detection
Pre-analytical factors influencing metanephrine levels
Posution, diet, medication, caffeine, exercise.
Supine and sitting much lower than standing
Supine much lower than sitting - recommendation to measure supine
Diet - catecholamine rich meal includes bananas, pineapples and walnuts
Coffee increases NMN but not MN
Exercise increases NMN and MN
True or false: metanephrine increases with age
FALSE - NMN increases with age
Expected effects of catecholamine rich meals on metanephrines
Increases in free 3MT, and all total metanephrines measurements. HOWEVER free NMN and MN do not increase. Nonetheless, best to take sample fasting.
Medications that falsely increase metanephrines
MAO-is (MN and NMNs)
TCAs (NMNs)
SNRIs (NMNs)
SSRIs (small effect on NMNs)
Sympathomimetics (dex, phentermine, meth etc) (MN and NMNs)
Alpha-2 blockers (phenoxybenzamine, mirtazapine) (NMN)
Beta-blockers (possible effect on MNs)
Levo-dopa (3MT)
What is the clonidine suppression test?
Clonidine (alpha 2 agonist) will suppress catecholamine release by sympathetic nerves in pts without PPGL
HEDT protocol for clonidine suppression test
Withhold interfering meds 5 days
Basline supine plasma mets x 2
Administer 300ug clonidine
Collect supine plasma mets at 3 hours
Normal result = normal normet +40% reduction in levels compared to baseline
Sensitivity and specificity of clonidine suppression test
Sens 94-97%, spec 100%
How does metabolic alkalosis develop in hyperaldosteronism?
Aldosterone increases potassium wasting through upregulation of basolateral Na-K ATPase and luminal K channels in the DCT. The intercalated A cells reabsorb K (to ameliorate hypokalaemia) in exchange for H+. Excretion of H+ results in alkalosis.
Causes of primary hyperaldosteronism
Aldosterone producing adenoma
Bilateral adrenal hyperplasia
Aldosterone producing carcinoma
Glucocorticoid-remediable hyperaldosteronism
Ectopic secretion from ovary or kidney
Causes of secondary hyperaldosteronism
With htn:
1. Renovascular
2. Reninoma
3. Phaeochromocytoma
With hypotension:
1. Volume depletion
2. Compensatory (nephrotic syndrome, cirrhosis, heart failure)
3. Bartter’s
4. Gitelman’s
Physiological
1. Pregnancy
2. Dehydration
Pharmacological
1. Diuretics
2. Vasosilators
Your lab’s method for salivary cortisol
Competitive electrochemiluminescent immunoassay. Ruthenium-labelled cortisol derivative competes with cortisol in the sample for binding to cortisol-specific biotinylated antibody. After incubation, streptavidin coated microparticles are added. Mixture aspirated and microparticles captured magnetically by the electrode. Unbound sample washed away. Voltage applied via electrode, causing ruthenium to chemiluminesce. Signal detected by photomultipler and is inversely proportional to concentration of cortisol in sample.
Effect of carbamazepine on dexamethasone suppression test?
CYP3A4 inducers such as carbamazepine may induce dexamethasone metabolism, as a result, less suppression of ACTH and cortisol occurs.
What is pro-renin?
Inactive precursor of renin - pro-segment masks active site of renin in closed conformation. In open conformation, pro-renin has same activity as renin.
Where is pro-renin produced?
Kidney (50%)
Adrenal glands
Gonads
Placenta
Retina
How and where is renin produced?
The only site of conversion of pro-renin to renin is the renal juxtaglomerular cells.
What is cryoactivation?
Unfolding of prorenin to open (active) conformation at temperatures < 37degC and low pH. There may also be subsequent cleavage by plasma proteases. Refolding occurs if the temperature is increased to back 37 or pH returned to physiological level.
Explain the difference in susceptibility of renin activity assays and concentration assays to cryoactivation
Incubation of sample at 37 deg C is required for activity assays, returning open pro-renin to the closed inactive form, more accurately reflecting physiological levels of renin activity. Immunoassays are more susceptible to cryoactivation effect because antibodies may “lock” prorenin into the open form and detect prorenin as renin.
Different types of plasma renin activity assays
- Endogenous angiotensinogen
- Exogenous angiotensinogen (sheep)
- Total renin activity assay (acidify to activate prorenin to renin, then add sheep angiontensinogen, OR, incubate with trypsinogen and use endogenous angiotensinogen)
Advantages of renin immunoassay
- Greater specificity due to recognition by two antibodies (sandwich design)
- Lower detection limits - add antibodies in excess to rapidly and completely detect all renin
Types of cortisol assay
Definitive: IDMS
Reference: LCMSMS
Routine:
1. Total serum cortisol - competitive immunoassay
2. Urine free cortisol - direct competitive immunoassay, extraction prior to immunoassay, HPLC (photodiode array), LCMSMS
3. Salivary cortisol - competitive immunoassay, LCMSMS
Methods of displacement of cortisol in routine direct competitive immunoassays
- 8-Anilino-Napthyl-Sulphonic acid (ANS; ANSA)
- Salicylate
- Danazol
Still overestimate cortisol in patients with high CBG (OCP, pregnancy)
