Dialyzable free cortisol after stimulation with Synacthen

OBJECTIVES: To compare the changes in free vs. total serum cortisol concentrations after acute stimulation of the adrenal cortex. DESIGN AND METHODS: Paired serum samples of ten individuals taken immediately before and 1 h after stimulation with 250 microg ACTH (1-24) (Synacthen) given i.v. were analyzed. Total cortisol was quantified using liquid chromatography tandem-mass spectrometry with an online sample extraction system and tri-deuterated cortisol as the internal standard. Free cortisol was measured with the same method after equilibrium dialysis. Concentrations of the corticosteroid-binding globulin (CBG) were determined by radioimmuno assay. RESULTS: Total cortisol increased by a mean of 106% (mean basal cortisol 312 nmol/L (SD 140 nmol/L), stimulated 686 nmol/L (SD 163 nmol/L); p < 0.001, paired t-test for differences); no significant change of CBG concentrations was found (874 nmol/L (SD 179 nmol/L) before stimulation, 869 nmol/L (SD 225 nmol/L) after stimulation). The mean increase of free cortisol was 263% (mean basal free cortisol 20.3 nmol/L (SD 13.2 nmol/L), stimulated 73.8 nmol/L (SD 26.7 nmol/L); p < 0.001) and thus substantially more pronounced compared to the increase of total cortisol (p < 0.01). The ratio of free to total serum cortisol was significantly increased after stimulation (6.1% (SD 1.7%) before stimulation, 10.6% (SD 1.9%) after stimulation; p < 0.001). CONCLUSIONS: After acute neuroendocrine stimulation of the adrenal cortex the relative increase of free bioactive cortisol concentrations is substantially more pronounced than the increase of total cortisol concentrations.