Clinical and Technical Aspects in Free Cortisol Measurement

Abstract

Accurate measurement of cortisol is critical in adrenal insufficiency as it reduces the risk associated with misdiagnosis and supports the optimization of stress dose. Comprehensive assays have been developed to determine the levels of bioactive free cortisol and their clinical and analytical efficacies have been extensively discussed because the level of total cortisol is affected by changes in the structure or circulating levels of corticoid-binding globulin and albumin, which are the main reservoirs of cortisol in the human body. Antibody-based immunoassays are routinely used in clinical laboratories; however, the lack of molecular specificity in cortisol assessment limits their applicability to characterize adrenocortical function. Improved specificity and sensitivity can be achieved by mass spectrometry coupled with chromatographic separation methods, which is a cutting-edge technology to measure individual as well as a panel of steroids in a single analytical run. The purpose of this review is to introduce recent advances in free cortisol measurement from the perspectives of clinical specimens and issues associated with prospective analytical technologies.

Keywords: Adrenal insufficiency; Cortisol; Immunoassay; Mass spectrometry; Saliva.

Fig. 1.

Distribution of cortisol in human blood. Corticoid-binding globulin (CBG) and albumin are main reservoirs of cortisol, binding 80%–90% and 10%–15% of total cortisol, respectively. Approximately 3%–5% of total cortisol is present as biologically active cortisol. States of altered CBG and albumin concentrations may lead to misrepresentation of cortisol action based on total cortisol levels.

Fig. 2.

A schematic diagram of free cortisol analysis in serum. Zinc sulfate is used to precipitate large proteins in serum and an aqueous buffer exchange is then performed to further precipitate the remaining small proteins and zinc ions in sample solution. Samples are vortexed and centrifuged before analysis using mass spectrometry or immunoassay, and only the supernatant is used.