Free and Total Plasma Cortisol Measured by Immunoassay and Mass Spectrometry Following ACTH1–24 Stimulation in the Assessment of Pituitary Patients

Abstract

Measurement of plasma cortisol by immunoassay after ACTH₁₋₂₄ stimulation is used to assess the hypothalamic-pituitary-adrenal (HPA) axis. Liquid chromatography-tandem mass spectrometry (LCMS) has greater analytical specificity than immunoassay and equilibrium dialysis allows measurement of free plasma cortisol. We investigated the use of measuring total and free plasma cortisol by LCMS and total cortisol by immunoassay during an ACTH₁₋₂₄ stimulation test to define HPA status in pituitary patients. This was a case control study conducted in a clinical research facility. We studied 60 controls and 21 patients with pituitary disease in whom HPA sufficiency (n = 8) or deficiency (n = 13) had been previously defined. Participants underwent 1 μg ACTH(1-24) intravenous and 250 μg ACTH₁₋₂₄ intramuscular ACTH₁₋₂₄ stimulation tests. Concordance of ACTH₁₋₂₄-stimulated total and free plasma cortisol with previous HPA assessment. Total cortisol was 12% lower when measured by immunoassay than by LCMS. Female sex and older age were positively correlated with ACTH₁₋₂₄-stimulated total and free cortisol, respectively. Measurements of total cortisol by immunoassay and LCMS and free cortisol 30 minutes after 1 μg and 30 and 60 minutes after 250 μg ACTH₁₋₂₄ were concordant with previous HPA axis assessment in most pituitary patients. However, free cortisol had greater separation from the diagnostic cutoff than total cortisol. Categorization of HPA status by immunoassay and LCMS after ACTH₁₋₂₄ stimulation was concordant with previous assessment in most pituitary patients. Free cortisol may have greater clinical use in patients near the diagnostic threshold.