The diagnostic suspicion of corticosurrenal disorders is based on a careful personal and family history and a careful physical examination. Laboratory tests and imaging play a central role in confirming the diagnostic hypothesis.
In particular, laboratory investigations include plasma and urinary assays of hormones and their metabolites, both under basal conditions and following stimulation or inhibition.
Basal Measurements
Blood
Cortisol
Ninety percent of cortisol in the blood circulates bound to proteins, and the remaining 10% is free; therefore, the evaluation of cortisolemia is influenced by the concentrations of its transport proteins (CBG and albumin); alterations of the transport proteins, both physiological (pregnancy) and pathological (infections in the acute phase, hepatic cirrhosis, nephrotic syndrome) can determine false cortisolemia values. In these cases, the free cortisol index is calculated rather than evaluating the total cortisolemia. This involves the evaluation of the ratio between total serum cortisol and CBG. Other formulas are available that consider not only CBG but also albumin. However, they are not yet used in clinical practice. Moreover, since cortisol has a circadian secretion, it is necessary to define the sampling time; it is generally performed between 8 and 9 a.m., when hormone concentration reaches its peak.
In normal subjects, serum cortisol concentrations are highest in the early morning (approximately 6:00 a.m.), ranging from 10 to 20 μg/dL. Serum cortisol concentrations range from 3 to 10 μg/dL at 4:00 p.m.; concentrations are lowest, less than 5 μg/dL, one hour after the usual sleep period.
Potassium
To increase the diagnostic accuracy of plasma potassium levels, increase the dietary sodium intake (2–3 g/day for at least 5 days) to identify latent hypokalemia. In addition, diuretic therapy or therapy with ACE inhibitors should be suspended for at least 3 weeks because it may give false positives. Normal potassium values are 3.5–4.5 mEq/L.
Aldosterone and Renin
Due to the circadian nature of aldosterone secretion, a single blood collection is not sufficient for measuring aldosterone and renin. In addition, aldosterone levels vary with body position (they increase with standing) and diet (with excessive potassium intake). Therefore, the patient should maintain the standing or supine position for at least 15–30 minutes before blood sampling. Normal plasma aldosterone values are 5–10 ng/dL in clinostat and <20 ng/ dL in orthostatic.
Two techniques exist for determining renin: measurement of plasma renin activity (PRA) and direct plasma renin assay. PRA is based on the principle that renin mediates the activation of angiotensinogen to angiotensin I and is performed as follows:
1. Prepare two tubes with the same amount of plasma to be tested for PRA.
2. The two tubes are incubated for one hour but at different temperatures: the first at 37 °C and the second at 4 °C.
3. At the end of the incubation, measure the amount of angiotensin I present in the two tubes. The PRA is the difference between the amount of angiotensin I measured in the first tube and the amount of angiotensin I measured in the second tube.
Values are expressed as angiotensin I (in ng) produced per mL of solution in one hour. In patients with optimal sodium intake (2 g/day), normal PRA values are 0.5–2.5 ng/mL/h in clinostat and 2–4 ng/mL/h in orthostatic. This is the most commonly used method.
Alternatively, the concentration of active renin, which has less inter-laboratory variability than PRA, can be measured directly in plasma. However, renin is labile, so special care must be taken when preparing the sample; blood must be collected in a pre-cooled tube, the sample must be centrifuged at 4 °C, and then the plasma quickly separated and frozen at −20 °C until the assay is performed.
In either case, sampling should be performed in the ortho static position (standing for at least two hours prior to sampling) or in the clinostat position (supine for at least two hours prior to sampling).
ACTH
Basal ACTH secretion shows a circadian rhythm with lower levels in the evening. Sampling for measurement of plasma levels should be performed in the morning, between 8 and 10 a.m. The ACTH assay is important in adrenal insufficiency because it allows differential diagnosis between primary and secondary forms.
DHEA Sulfate
It is secreted primarily by the adrenals and only in small amounts by the gonads. Thus, DHEA sulfate is a helpful indicator of adrenal androgen secretion.
Urinary Measurements
Free Cortisol
Measurement of free urinary cortisol is helpful in the assessment of adrenal function. Since free cortisol is filtered at the glomerular level and excreted by the kidney, the cortisol measurement in the 24-hour urine represents the free portion of serum cortisol and, at the same time, its “integrated” measurement, unaffected by variations due to the circadian rhythm. Immunometric methods perform the assay. One of the main limitations of the assay is the inadequacy of the 24-hour urine collection. It is, therefore, advisable to perform the urine creatinine measurement to verify the validity of the collection. Since cortisol secretion is subject to daily fluctuations, collecting 24-hour urine for 2–3 days is recommended.
Salivary Assays
Hormones are present in saliva in a free form and their concentrations correlate significantly with their free plasma fraction.
Saliva is usually collected by inserting a swab into the patient’s mouth and waiting a few minutes until it is completely soaked in saliva; usually, two samples are collected.
Cortisol
Salivary cortisol represents a surrogate marker of free cortisol, in equilibrium with free serum cortisol. Furthermore, it is not affected by saliva production rate or transport proteins. The assay is performed by immunometric techniques or by mass spectrometry.
The determination of cortisol on a saliva sample collected around midnight (nocturnal salivary cortisol [NSC]) represents an important screening test for Cushing’s syndrome, characterized by increased salivary and plasma cortisol levels at night. Salivary cortisol has the advantage that it can be collected at home by self-sampling and, being stable at room temperature, taken the following morning to a laboratory for analysis. However, it must be taken into account that the immunological assay could lead to false positives due to cross-reactivity with cortisone that salivary glands convert from cortisol by the enzyme 11-β-hydroxysteroid dehydrogenase type 2.
One of the main advantages of salivary cortisol is that levels tend to be altered when urinary-free cortisol is normal or only modestly increased in patients with Cushing’s syndrome.
Salivary cortisol determination performed in the morning is a helpful screening test for adrenal insufficiency; values <1.5 μg/L is strongly indicative of the disease, while values >6.2 μg/L exclude adrenal insufficiency.
Dynamic Investigations
Dynamic laboratory investigations are aimed at assessing the integrity and function of the hypothalamic pituitary adrenal (HPA) axis; a distinction is made between stimulation tests, which are useful in diagnosing conditions of hormone deficiency, and suppression tests, which are useful in documenting hypersecretion of adrenal hormones (Tables 1, 2, 3, 4, 5, and 6).
Table1. Rapid stimulation test with ACTH
Table2. Stimulus test with CRH
Table3. Insulin tolerance test (ITT)
Table4. Metyrapone test
Table5. Low-dose dexamethasone test
Table6. High-dose dexamethasone test
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