CushieBlog

Cushing’s syndrome (CS) is characterized by a state of hypercortisolism that can be detected and monitored by means of clinical laboratory tests, such as 24 h urinary freecortisol (UFC). UFC measurement constitutes one of the three first level recommended tests, along with overnight 1 mg dexamethasone suppression and late night salivary cortisol tests [1], [2].

UFC levels are in general highly variable, and at least two 24 h urine collections are necessary for screening/monitoring of CS [1], [2]. In addition to this, 24 h urine samples are often further required due to unexpected or biochemically inconsistent results. This makes the process even more tedious for the patient, and ultimately causes delays in CS diagnosis and management.

Such discordant results may derive from an undeclared use of exogenous glucocorticoids or analytical limitations, among other reasons. The latter occurs especially when UFC analyses are performed by immunoassays (IAs), due to their limited specificity.

Therefore, improvements in UFC tests concerning the analytical methodology, and the inclusion of complementary biomarkers that reinforce their clinical interpretation in the light of unexpected/inconsistent results, appear necessary.

Besides, the simultaneous monitoring of exogenous glucocorticoids in UFC analyses, which is not often considered in clinical practice, should be included.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been suggested as the most suitable alternative for UFC quantification [3], [4], [5], [6], [7], since it overcomes IAs analytical limitations. Besides, it also allows for the simultaneous monitoring of different analytes.

In the context of CS management, the simultaneous LC-MS/MS determination of UFC and cortisone, as well as the use of cortisol/cortisone ratio have been previously suggested [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19]. With regard to the monitoring of exogenous glucocorticoids, at our center (Hospital Universitario Son Espases, Palma, Spain), it would be of particular importance in the case of dexamethasone. This is because the UFC determination is often followed by an overnight 1 mg dexamethasone suppression test, and its intake may occur by mistake prior or during urine collection.

Despite their advantages, LC-MS/MS methods usually require time-consuming sample preparations, e.g. liquid-liquid extraction (LLE) protocols [20], thus not allowing to completely avoid using IAs in most clinical laboratories. In Spain, to the best of our knowledge, all hospitals in the public healthcare system still use IAs for UFC analysis. Mass spectrometry is only available at tertiary-care centers or academic hospitals, where is still used in combination with IAs to cope with the large volume of samples received on a daily basis. In this case, the use of complementary biomarkers in LC-MS/MS UFC analyses would be of particular interest, as discordant results may occur between methods due to IA analytical limitations.

For all these reasons, in the herein presented study, a novel, rapid and accurate LC-MS/MS method based on direct injection approach for the quantitation of UFC, cortisone, and dexamethasone was developed. Given the lack of standardization in reference ranges, appropriate population-based LC-MS/MS reference values were established.

Most research only focuses on the ability of cortisone and cortisol/cortisone ratio to discriminate ectopic ACTH production from other subtypes of CS [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19]. For this reason, this study assessed their suitability as complementary biomarkers, and therefore their ability to reinforce the clinical interpretation of UFC analyses.

To the best of our knowledge, they have not been previously assessed in the context of hypocortisolism. This would be of substantial importance in the follow-up of CS since adrenal insufficiency secondary to adrenalectomy/pituitary surgery or pharmacological treatment (e.g. ketoconazole, metyrapone) may occur. Therefore, such scenario was further considered.

Read more at https://www.sciencedirect.com/science/article/abs/pii/S0009898126000914