by Dr Federico Ponzetto

Cushing’s syndrome is related to high production of cortisol and, without timely treatment, it can lead to complications such as heart attacks, strokes and type 2 diabetes. The condition can be diagnosed using a dexamethasone suppression test, which involves tracking the effects of this glucocorticoid medication on cortisol levels. Since the absorption of dexamethasone depends on several factors – such as metabolism and reactions with other drugs – it is useful to measure its concentration concurrently with cortisol to avoid false positives. A group at the University of Turin led by Professor Giulio Mengozzi in the Department of Medical Sciences has developed a liquid chromatography–tandem mass spectrometry method for simultaneous quantification of cortisol, cortisone, dexamethasone and six additional exogenous corticosteroids, enabling a more accurate diagnosis of Cushing’s syndrome.

Cushing’s syndrome

Cushing’s syndrome is characterized by abnormally high cortisol production over a long period, and is known to affect adults between the ages of 30 and 50 years, predominantly occurring in females [1]. The disease can be endogenous – where the issue originates from something inside the body – but is more commonly caused by external factors, such as glucocorticoid medications.

Cushing’s syndrome is associated with many visible symptoms, such as weight gain, increased fat around the base of neck, fatty hump between the shoulders, ‘moon face’ and easy bruising. However, not everyone with Cushing’s syndrome exhibits these symptoms, making the condition difficult to diagnose. If left untreated, it can cause a range of serious complications, including heart attack and stroke, blood clots in legs and lungs, increased risk of infections, memory loss and type 2 diabetes.

Dexamethasone testing

Cushing’s syndrome can be diagnosed through a dexamethasone suppression test (DST), which measures the response of the adrenal glands to adrenocorticotropic hormone (ACTH) that is secreted from the pituitary gland and normally regulates the level of cortisol in the blood plasma. ACTH stimulates the adrenal cortex to produce cortisol and, when plasma cortisol levels increase, the ACTH secretion is suppressed. As dexamethasone is a synthetic steroid similar to cortisol, administering this drug should lead to a reduction in ACTH levels.

There are two types of DSTs – low- and high-dose options – both of which can be performed either overnight or over a two-day period. Low-dose DSTs (LDDSTs) are used to obtain an initial Cushing’s syndrome diagnosis and, if the result is positive, this is followed by high-dose DST (HDDST) that can help categorize the disease as ACTH dependent or independent. These tests are usually carried out in the following manner [2].

Cortisol is a steroid hormone of the glucocorticoid class made by the adrenal glands (Shutterstock.com)

LDDST

– Overnight protocol: 1 mg of dexamethasone is administered at 11.00 pm, and the serum cortisol levels are measured at 8.00 am the following morning.
– Two-day protocol: serum cortisol levels are measured at 8.00 am and 0.5 mg of dexamethasone is administered every six hours (9.00 am, 3.00 pm, 9.00 pm, 3.00 am) for two days, totalling 4 mg. Serum cortisol levels are then measured at 9.00 am, six hours after the last dose has been delivered.

HDDST

– Overnight protocol: baseline serum cortisol or 24-hour urinary free cortisol (UFC) is measured in the morning, and 8 mg of dexamethasone is given at 11.00 pm. Cortisol level in blood is then measured at 8.00 am the following morning.
– Two-day protocol: Baseline serum cortisol or 24-hour UFC is measured at 8.00 am; 2 mg of dexamethasone is administered every six hours (9.00 am, 3.00 pm, 9.00 pm, 3.00 am) for two days, totalling 16 mg, in tandem with collection of urine for UFC measurements. Serum cortisol levels are measured at 9.00 am, six hours after the last dose has been delivered.

Patients whose pituitary glands produce too much ACTH will have an abnormal response to the low-dose test, but a normal reaction to the high-dose test. For LDDST, the cortisol levels should decrease in response to dexamethasone administration, and a value below 18 ng/mL is the recommended cut-off value to separate a healthy from an unhealthy reaction. In the case of HDDST, a reduction in UFC or serum cortisol greater than 50% indicates that the patient has ACTH-dependent Cushing’s syndrome. This rule applies to both the overnight LDDST and two-day HDDST approaches.

Measuring cortisol levels

Chemiluminescence immunoassay (CLIA) is the most common method for measuring cortisol and other steroids, as it is easy to use, has a workflow that can be fully automated, and offers good sensitivity. Unfortunately, this technique has several drawbacks, the main one being cross-reactivity that can lead to an over-estimation of target analyte levels. Other flaws are the lack of standardization between different kits on the market, and the fact that it can only measure one analyte per analysis. This can be problematic as several studies suggest that measuring dexamethasone in tandem with cortisol can help to reduce the number of false-positive results for DSTs [3,4] and improve interpretability [5] – as it is hard to predict how a patient’s body will react to the synthetic steroid. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) has become a popular alternative to CLIA for DSTs, owing to its ability to measure several analytes at once and its much higher specificity. Target analytes are separated through LC, and their concentrations are measured using MS. Triple quadrupole MS set-ups are commonly used for this purpose, providing not only the ability to measure several analytes at once, but also higher accuracy and sensitivity than CLIA.

Increased ease-of-use and accuracy

A group in the Division of Endocrinology, Diabetes and Metabolism at the University of Turin has created an LC-MS/MS method for simultaneous quantification of cortisol, cortisone, dexamethasone and six additional exogenous corticosteroids in serum/plasma samples [6]. The method can be easily implemented in any clinical laboratory with a mass spectrometer, and has proven useful for DSTs, enabling accurate measurements of the target analytes in a single chromatographic run (Fig. 1). The method is outlined on the next page.

Sample preparation (1 hour)

1. Dilute 200 μL of the serum/plasma sample with 200 μL of water.
2. Perform supported liquid extraction, transferring 400 μL of sample manually to a microplate.
3. Apply positive pressure using Tecan Resolvex® A200 automated positive pressure processor.
4. Elute with 700 μL of methyl tert-butyl ether.
5. Evaporate and reconstitute in H2O/MeOH (1:1, v/v).
6. Agitate.

LC-MS/MS analysis (10 minutes)

• LC column: C18 (100 × 2.1 mm, 1.7 μm)
• flow rate: 400 μL/min
• temperature: 30 °C
• injection volume: 20 μL
• mobile phase A: H2O + 0.2 mM ammonium fluoride
• mobile phase B: acetonitrile
• elution programme: see Table 1.

The results showed good correlation with measurements performed using a commercially available CE IVD-marked Steroid Panel LC-MS kit* (Tecan). This kit can measure dexamethasone, cortisol and cortisone simultaneously, and includes all essential components for easy implementation, such as calibrators and controls. The samples are prepared using solid-phase extraction (SPE), which can be performed semi-automatically on a Resolvex® A200 positive pressure processor (Tecan). Thanks to the effective SPE process, the kit can also measure 15 additional steroids in the core steroid metabolism pathway.

Figure 1. Example chromatogram of the Steroid Panel LC-MS internal standard – run 1
ESI, electrospray ionization; 1, aldosterone; 2, cortisone; 3, dehydro­-epiandrosterone sulfate; 4, cortisol; 5, 21-deoxycortisol; 6, corticosterone; 7, dexamethasone; 8, 11-deoxycortisol; 9, androstenedione; 10, 11-deoxy-corticosterone; 11, testosterone; 12, dehydroepiandrosterone; 13, 17-hydroxyprogesterone; 14, dihydrotestosterone; 15, progesterone.

Summary

Cushing’s syndrome is a serious condition that should be diagnosed as soon as possible to avoid life-threatening complications. Reducing the number of false positives in DSTs requires simultaneous measurement of both cortisol and dexamethasone levels, which can be performed successfully using LC-MS/MS. The LC-MS/MS method described in this article makes it possible to track several analytes simultaneously – including cortisol, cortisone and dexamethasone – in serum or plasma. Implementing this analytical approach will offer clinical laboratories a straightforward way to perform DSTs, and the commercially available kit will help to ensure reliable and reproducible results.

Table 1. LC gradient elution programme

* In USA: for research use only. Not for use in diagnostic procedures. Product availability and regulatory status may vary from country to country. Consult with your Tecan associate for further information.

References

1. Cushing’s syndrome [website]. National Institute of Diabetes and Digestive and Kidney Diseases 2018 (https://www.niddk.nih.gov/health-information/endocrine-diseases/cushings-syndrome).
2. Dogra P, Vijayashankar NP. Dexamethasone suppression test. StatPearls 2022, 8 August
(https://www.ncbi.nlm.nih.gov/books/NBK542317).
3. Ceccato F, Artusi C, Barbot M, et al. Dexamethasone measurement during low-dose suppression test for suspected hypercortisolism: threshold development with and validation. J Endocrinol Invest 2020;43(8):1105–1113. doi: 10.1007/s40618-020-01197-6.
4. Roper SM. Yield of serum dexamethasone measurement for reducing false-positive results of low-dose dexamethasone suppression testing. J Appl Lab Med 2021;6(2):480–485. doi: 10.1093/jalm/jfaa193.
5. Fleseriu M, Auchus R, Bancos I, et al. Consensus on diagnosis and management of Cushing’s disease: a guideline update. Lancet Diabetes Endocrinol 2021;9(12):847–875. doi: 10.1016/S2213-8587(21)00235-7.
6. Ponzetto F, Parasiliti-Caprino M, Settanni F, et al. Simultaneous measurement of cortisol, cortisone, dexamethasone and additional exogenous corticosteroids by rapid and sensitive LC-MS/MS analysis. Molecules 2022;28(1):248. doi: 10.3390/molecules28010248.

The author

Federico Ponzetto PhD
Division of Endocrinology, Diabetology and Metabolism, Department of Medical Sciences, University of Turin, Turin, Italy

E-mail: federico.ponzetto@unito.it